Calanolide and related antiretroviral compounds, compositions, and uses thereof

ABSTRACT

The present invention provides novel antiviral compounds, refered to as calanolides, related compounds, and their derivatives, which may be isolated from plants, or derived from compounds from plants, of the genus Calophyllum in accordance with the present inventive method. The compounds and their derivatives may be used alone or in combination with other antiviral agents in compositions, such as pharmaceutical compositions, to inhibit the growth or replication of a virus, such as a retrovirus, in particular a human immunodeficiency virus, specifically HIV-1 or HIV-2.

This patent application is a continuation-in-part of copending U.S.patent application Ser. No. 07/861,249 filed on Mar. 31, 1992.

TECHNICAL FIELD OF THE INVENTION

This invention relates to antiviral compounds, in particular antiviralcompounds isolated from, or derived from compounds isolated from, plantsof the genus Calophyllum, specifically compounds referred to ascalanolides. This invention also relates to methods of isolatingantiviral compounds from Calophyllum plants, compositions comprisingcalanolides, related compounds, and derivatives thereof, and methods ofusing the compositions in clinical applications, such as antiviraltherapy and the prevention of viral infection.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is a very serious disease,reported cases of which have increased dramatically within the pastseveral years. Estimates of reported cases in the very near future alsocontinue to rise dramatically. Consequently, there is a great effort todevelop drugs and vaccines to combat AIDS.

The AIDS virus was first identified in 1983. It has been known byseveral names and acronyms. It is the third known T-lymphocyte virus(HTLV-III), and it has the capacity to replicate within cells of theimmune system, causing profound cell destruction. The AIDS virus is aretrovirus, a virus that uses reverse transcriptase during replication.This particular retrovirus is also known as lymphadenopathy-associatedvirus (LAV), AIDS-related virus (ARV) and, most recently, as humanimmunodeficiency virus (HIV). Two distinct types of HIV have beendescribed to date, namely HIV-1 and HIV-2. The acronym HIV will be usedherein to refer to HIV viruses generically.

Specifically, HIV is known to exert a profound cytopathic effect on theCD4+ helper/inducer T-cells, thereby severely compromising the immunesystem. HIV infection also results in neurological deterioration and,ultimately, in the death of the infected individual.

The field of viral chemotherapeutics has developed in response to theneed for agents effective against retroviruses, in particular HIV. Thereare many ways in which an agent can exhibit anti-retroviral activity.For example, HIV requires at least four viral proteins for replication:reverse transcriptase (RT), protease (PR), transactivator protein (TAT),and regulator of virion-protein expression (REV). Accordingly, viralreplication could theoretically be inhibited through inhibition of anyone or all of the proteins involved in viral replication.

Anti-retroviral agents, such as AZT and ddC, are known to inhibit RT.There also exist anti-viral agents that inhibit TAT.

Nucleoside derivatives, such as AZT, are the only clinically activeagents that are currently available for antiviral therapy. Although veryuseful, the utility of AZT and related compounds is limited by toxicityand insufficient therapeutic indices for fully adequate therapy.

Synthetic peptides also are being developed for potential use asinhibitors of the retroviral protease in the treatment of AIDS. Althoughthese inhibitors are effective in preventing the retroviral proteasefrom functioning, the inhibitors suffer from some distinctdisadvantages. First of all, since the active site of the protease ishindered, i.e., has reduced accessibility as compared to the remainderof the protease, the ability of the inhibitors to access and bind in theactive site of the protease is impaired. Secondly, the peptideinhibitors that bind to the active site of the protease are generallypoorly soluble in water, causing distinct problems in drug delivery.

Therefore, new classes of antiviral agents to be used alone or incombination with AZT and/or other agents are urgently needed foreffective antiviral therapy against HIV. New agents, which may be usedto prevent HIV infection, are also important.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel antiviralcompounds, in particular antiviral compounds isolated from plants of thegenus Calophyllum, specifically compounds referred to as calanolides,related compounds, and derivatives thereof.

It is another object of the present invention to provide a method ofisolating novel antiviral compounds, specifically calanolides, relatedcompounds, and derivatives thereof, from plants of the genusCalophyllum, more specifically from Calophyllum lanigerum Miq., var.austrocoriaceum (T. C. Whitmore) P. F. Stevens, from Calophyllumteysmannii Miq. var inophylloide (King) P. F. Stevens.

It is still another object of the present invention to provide acomposition, in particular a pharmaceutical composition, which inhibitsthe growth or replication of a virus, such as a retrovirus, inparticular a human immunodeficiency virus, specifically HIV-1 or HIV-2.

It is an additional object of the present invention to provide acomposition, in particular a pharmaceutical composition, which preventsinfection of an animal, in particular a human, with a virus, such as aretrovirus, in particular a human immunodeficiency virus, specificallyHIV-1 or HIV-2.

Yet another object of the present invention is to provide a method oftreating an animal, in particular a human, infected with a virus, suchas a retrovirus, in particular a human immunodeficiency virus,specifically HIV-1 or HIV-2.

A further object of the present invention is to provide a method oftreating an animal, in particular a human, to prevent infection with avirus, such as a retrovirus, in particular a human immunodeficiencyvirus, specifically HIV-1 or HIV-2.

These and other objects of the present invention, as well as additionalinventive features, will become apparent from the description herein.

The present invention provides novel antiviral compounds, in particularantiviral compounds isolated from, as well as derivatives of compoundsisolated from, plants of the genus Calophyllum (particularly,Calophyllum lanigerum var. austrocoriaceum and Calophyllum teysmanniivar. inophylloide), specifically compounds referred to as calanolides,related compounds, and derivatives thereof in substantially pure form.The present invention also provides for a method of isolating andpurifying calanolides and related antiviral compounds from Calophyllumplants, in particular from Calophyllum lanigerum Miq., and fromCalophyllum teysmannii Miq. The isolated and derived compounds may beused in a composition, such as a pharmaceutical composition, which mayadditionally comprise one or more other antiviral agents. Such acomposition has been found to inhibit the growth or replication of avirus, in particular a retrovirus, specifically a human immunodeficiencyvirus, such as HIV-1 or HIV-2. The composition, therefore, is expectedto have utility in the therapeutic treatment of an animal, such as ahuman, infected with a virus, particularly a retrovirus, specifically ahuman immunodeficiency virus, such as HIV-1 or HIV-2, and in theprophylactic treatment of an animal, such as a human, to prevent viralinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structures of calanolides and related derivatives(compounds 1-8) isolated from Calophyllum lanigerum var.austrocoriaceum. Compound 1 is calanolide A, and compound 4 iscalanolide B.

FIG. 2 illustrates previously known structures (compounds 9-4) reportedfrom other sources.

FIG. 3 shows the ¹ H-NMR values (Δδ=δ_(S) -δ_(R) in Hertz at 500 MHz)for (R)- and (S)-MTPA esters of calanolide A (1) and calanolide B (4)used in the determination of the absolute configuration of calanolides Aand B.

FIG. 4, A-D, shows an example of anti-HIV-1 activity of a calanolide.FIGS. 4A, 4B, and 4C show the effects of a range of concentrations ofcalanolide A upon uninfected CEM-SS cells (o) and upon CEM-SS cellsinfected with HIV-1 (), as determined after 6 days in culture; FIG. 4Adepicts the relative numbers of viable CEM-SS cells, as assessed by theBCECF assay; FIG. 4B depicts the relative DNA content of the respectivecultures; FIG. 4C depicts the relative numbers of viable CEM-SS cells,as assessed by the XTT assay. FIG. 4D shows the effects of a range ofconcentrations of calanolide A upon indices of infectious virus or viralreplication; these indices include viral reverse transcriptase (▴),viral core protein p24 (♦) and syncytium-forming units (▪). In FIGS. 4A,4B, and 4C, the data points are represented as the percent of theuninfected, non-drug treated control values. In FIG. 4D the data pointsare represented as the percent infected, non-drug treated controlvalues.

FIGS. 5A and 5B more generally illustrate calanolides and derivativesthereof (series 1) and 7,8-dihydrocalanolides and derivatives thereof(series 2), respectively, wherein R¹ is C₁ -C₆ alkyl or aryl; R² is OH,OH, OR³, OR³, O₂ CR³, O₂ CR³, O₃ SR³, or O₃ SR³, wherein R³ is C₁ -C₆alkyl or aryl; and R⁴ and R⁵ are the same or different and are each CH₃or CH₃. (The symbol indicates a bond that extends out of the plane ofthe paper toward the reader, whereas the symbol indicates a bond thatextends below the plane of the paper away from the reader.)

FIGS. 6, A-D, shows examples of antiviral 7,8-dihydrocalanolides andrelated antiviral 7,8-dihydro compounds.

By convention with the chemical literature, when only the symbols or areshown within individual chemical structures (e.g., as in FIGS. 1 and 6),they are assumed to be equivalent to CH₃ and CH₃, respectively.

FIG. 7, A-H, illustrates anti-HIV-1 activities of calanolide A,7,8-dihydrocalanolide A, calanolide B, 7,8-dihydrocalanolide B,costatolide, 7,8-dihydrocostatolide, soulattrolide, and7,8-dihydrosoulattrolide, respectively. The effects of a range ofconcentrations of each compound upon cellular viability was assessedusing the XTT assay after 6 days in culture in uninfected CEM-SS cells(o) and in CEM-SS cells infected () with HIV-1. The data points arerepresented as the percent of the uninfected, non-drug treated controlvalues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antiviral compounds (hereinafterreferred to as "calanolides"), related compounds, and derivativesthereof, in substantially pure form and having the structures and names:##STR1## To the extent the aforesaid compound names are used in thecontext of describing and claiming the present invention herein, thosecompound names have reference to the corresponding chemical structuresset out immediately above. The antiviral calanolides, related compounds,and derivatives thereof may be described generically as having thestructures: ##STR2## wherein R¹ is C₁ -C₆ alkyl or aryl; R² is OH, OH,OR³, OR³, O₂ CR³, O₂ CR³, O₃ SR³, or O₃ SR³, wherein R³ is C₁ -C₆ alkylor aryl; R⁴ and R⁵ are the same or different and are each CH₃ or CH₃.While the aryl group may be any suitable aryl substituent, the aryl ispreferably a C₆ -C₁₄ ring structure, most preferably phenyl.

The present invention also provides a method of isolating and purifyingcalanolides and related antiviral compounds and derivatives from plantsof the genus Calophyllum, which comprises the steps of:

a) extracting plant material with organic solvents to obtain a crudeextract having antiviral activity;

b) solvent-solvent partitioning the crude extract, as necessary, toconcentrate an antiviral compound in a non-polar fraction;

c) subjecting the crude extract or the non-polar fraction to gelpermeation or vacuum liquid chromatography as necessary to furtherconcentrate the antiviral compound; and,

d) isolating and purifying the antiviral compound by HPLC on silica geland C₁₈ reversed-phase columns.

This method is used in conjunction with an antiviral assay to identifythe antiviral fractions obtained in the various process steps and may beused to obtain antiviral calanolides, related antiviral compounds, andantiviral derivatives from plant material consisting of leaves, stems,twigs, fruits, flowers, wood, bark, or roots of the said Calophyllumplants. Such antiviral compounds may also be obtained by this method,wherein steps b) and c) may be deleted, from the latex harvestednondestructively from Calophyllum plants.

The antiviral calanolides, related antiviral compounds, and antiviralderivatives thereof, obtained in accordance with the present inventivemethod, may be used alone or in combination with other antiviral agentsin compositions, such as pharmaceutical compositions, to inhibit thegrowth or replication of a virus, such as a retrovirus, in particular ahuman immunodeficiency virus, specifically HIV-1 or HIV-2. It isexpected that such compositions will have utility in the therapeutictreatment of an animal, in particular a human, infected with one or moreof the above-cited viruses and in the prophylactic treatment of ananimal, in particular a human, who is at risk for infection with one ormore of the same viruses.

Prior to the discovery on which this invention is based, antiviralcalanolides and derivatives thereof had not been isolated or described.Methods for isolating such compounds had not been determined. Theisolation and chemical structures of related compounds, costatolide anddihydrocostatolide (Stout, G. H., J. Org. Chem., 29, 3604-3609 (1964))and soulattrolide (Gunasekera, S. P., et al., J. Chem. Soc. Perkin I,1505-1511 (1977)) had previously been reported in the chemicalliterature; however, no antiviral activity was reported for thesecompounds. Accordingly, since the antiviral activity of none of thesecompounds was previously known, the potential use of these compounds incompositions, such as pharmaceutical compositions, in the therapeuticand prophylactic treatment of viral infections in animals, in particularhumans, had not been recognized.

General Information

An initial observation that led to the present invention was theantiviral activity of extracts from Calophyllum plants in an anti-HIVscreen. The screen is one that has been operated by the U.S. NationalCancer Institute since 1988 (see Boyd, M. R., in AIDS, Etiology,Diagnosis, Treatment and Prevention (DeVita V. T., Jr., Hellman S.,Rosenberg S. A., eds.), pp. 305-319 (Philadelphia: Lippincott, 1988)).

The plant family Guttiferae contains the genus Calophyllum, which iscomprised of approximately 187 different species. A brief description ofthe genus Calophyllum can be found in D. J. Mabberley, "The Plant Book",Cambridge, University Press, 1987, p. 92. The most updated taxonomicrevision of the Old World species of the genus has been published byStevens, P. F., J. Arnold Arbor., 61, 117-699 (1980). Of the 187 knownspecies, 179 are found in the Old World, from the shores of TropicalAfrica east to Tahiti in the Pacific, through Madagascar, Sri Lanka,Southeast Asia, New Guinea, and Northern Australia. The greater numberof species, however, occur in Indonesia, Malaysia, and the Philippines.

Previous phytochemical studies of the genus Calophyllum had revealed itto be a rich source of secondary metabolites. Xanthones (Dharmaratne, H.R. W., et al., Phytochemistry, 25, 1957-1959 (1986); Kumar, V.,Phytochemistry, 21, 807-809 (1982); Somanathan, R., et al., J. Chem.Soc. Perkin I, 2515-1517 (1974)), steroids (Gunasekera, S. P., et al.,J. Chem. Soc. Perkin I, 2215-2220 (1975)), triterpenes (Gunatilaka, A.A. L., et al., Phytochemistry, 23, 323-328 (1984); Dahanayake, M., etal., J. Chem. Soc. Perkin I, 2510-2514 (1974)), coumarins (Samaraweera,U. et al Tetrahedron Lett., 22, 5083-5086 (1981); Gautier, J., et al.,Tetrahedron Lett., 27, 2715-2718 (1972)), and benzopyrans (Stout, G. H.,J. Org. Chem., 33, 4185-4190 (1968)) are among the compounds reportedfrom Calophyllum species; however, no antiviral activity had beenpreviously associated with any compounds from this genus.

A specific bioassay-guided strategy was used to isolate, purify, andidentify the individual bioactive compounds from the extracts ofCalophyllum plants. In this strategy, decisions concerning the overallchemical isolation method to be applied, and the nature of theindividual steps therein, were determined by interpretation ofbiological testing data. The anti-HIV screening assay (Weislow, O. S.,et al., J. Natl. Cancer Inst., 81(8), 577-586 (1989)) used in thisprocess measured the degree of protection of human T-lymphoblastoidcells from the cytopathic effects of HIV. Fractions of the extracts wereprepared using a variety of chemical means and were tested blindly inthe primary screen. Active fractions were separated further, and theresulting subfractions were tested blindly in the screen. This processwas repeated as many times as necessary in order to obtain the active,i.e., antiviral, fraction(s) representing pure compound(s), which thencould be subjected to detailed chemical analysis and structureelucidation. In this manner, the new antiviral class of compoundsdescribed herein was discovered.

Although Calophyllum lanigerum var. austrocoriaceum and Calophyllumteysmannii var. inophylloide were used as the principal sources ofcalanolides and related compounds in the present invention, it will beappreciated that such compounds also may be obtained from other speciesof the same genus. For example, such other source species may includeCalophyllum lanigerum var. lanigerum, Calophyllum teysmannii var.teysmannii, and Calophyllum teysmannii var. bursiculum P. F. Stevens.

Taxonomy

Calophyllum lanigerum var. austrocoriaceum. The plant material utilizedin Example 2 herein specifically belongs to var. austrocoriaceum ofCalophyllum lanigerumand was collected by J. S. Burley & B. Lee on Oct.18, 1987 in Setunggang swamp forest near Batang Kayan River, of theMunicipality of Lundu, Sarawak (East Malaysia), at an altitude of 3 mabove sea level and at a latitude of 2° N and a longitude of 110° E,under a U.S. National Cancer Institute contract to D. D. Soejarto of theUniversity of Illinois at Chicago. This collection is documented byvoucher herbarium specimens Burley & Lee 351, in deposit at the U.S.National Herbarium of the Smithsonian Institution, Washington, D.C.Duplicates of this specimen are also in deposit at the Sarawak ForestHerbarium in Kuching (East Malaysia), the John G. Searle Herbarium ofthe Field Museum of Natural History, Chicago (Ill.), and the ArnoldArboretum Herbarium of Harvard University in Cambridge (Mass.). Theidentity of these specimens as Calophyllum lanigerum Miq. var.austrocoriaceum (T. C. Whitmore) P. F. Stevens was made by Dr. Peter F.Stevens, Arnold Arboretum of Harvard University, the taxonomicspecialist of the genus Calophyllum. Further taxonomic details of thespecific plant used in Example 2 are provided in Example 1 below.

Calophyllum teysmannii var. inophylloide. The plant material utilized inExample 5 herein specifically belongs to var. inophylloide ofCalophyllum teysmannii. In March 1992, as part of a field search in anattempt to recollect Calophyllum lanigerum var. austrocoriaceum, samplesof a Calophyllum species (Soejarto et al. 7605) from a kerangas forestnear Sampedi forest reserve, about 50 km west of Kuching, Sarawak, werecollected. Anti-HIV tests of extracts from this plant (leaf and twig;stem) showed activity. Dr. Peter F. Stevens of Harvard University, thespecialist of Calophyllum, identified this 7605 specimen as Calophyllumteysmannii Miq. var. inophylloide (King) P. F. Stevens.

In July 1992, as part of the continuing field search for Calophyllumlanigerum var. austrocoriaceum, latex samples of other Calophyllumspecies were collected, two of which belonging to Soejarto et al. 7853and 7854 trees also showed anti-HIV activity. The locality of thesetrees is Sampedi Forest Reserve kerangas forest, separated about 1 kmfrom the locality of 7605, at an altitude of between 30-60 m above sealevel. Both 7853 and 7854 trees were marked by an orange plastic ribbon,and numbered using an embossed aluminum plate, affixed to the trees witha nail.

On Jan. 7 and 8, 1993, voucher herbarium specimens and more latexsamples were recollected from both trees 7853 and 7854, with theassistance of staff of the Sarawak Forest Department. In addition,further search in the surrounding areas yielded four (4) more trees(Soejarto et al. 7899-7902) of the same species as 7853 and 7854, fromwhich latex samples were also collected. These trees were similarlymarked and numbered for future relocation.

When voucher herbarium specimens of Soejarto et al. 7605 were comparedwith those of 7853, 7854, and 7899-7902, it was apparent that they areall of the same species, namely, Calophyllum teysmannii Miq. var.inophylloide (King) P. F. Stevens. A duplicate set of voucher specimens7853 and 7854 was sent to Dr. Peter F. Stevens on Jan. 17, 1993; on Jan.19, 1993, Dr. Stevens confirmed the identity of 7853 and 7854 asCalophyllum teysmannii var. inophylloide. Further taxonomic details ofthe specific plants used in Examples 7 and 8 below are provided inExample 6 and Table 3 herein.

Isolation And Purification Of Calanolides And Related AntiviralCompounds From Calophyllum Plants

A variety of methods can be used for the chemical isolation ofcalanolides and related antiviral compounds from Calophyllum plants.Among these methods are extraction, solvent-solvent partitioning, flash-or vacuum-liquid chromatography, gel permeation chromatography, andHPLC, with a variety of bonded phases. The isolation of the pure activecompounds can be monitored by UV, TLC, and anti-HIV bioassay. Typicalisolation procedures are set forth below for illustrative purposes.

Antiviral calanolides and related antiviral compounds from Calophyllumplant parts. Approximately 0.2 kilogram of air-dried plant material, forexample, consisting of leaves, twigs, fruit or bark, is first ground toa fine powder and extracted with 1:1 MeOH-CH₂ Cl₂ ; this is followed bya second extraction with methanol. These organic extracts typicallyamount to a total of about 5-20% of the mass of the starting plantmaterial. The initial crude extract is dissolved in 4:1 MeOH-H₂ O andextracted three times with CCl₄. The concentrated CCl₄ phase isfractionated by either gel permeation on Bio-Beads S-X4 (Bio-RadLaboratories, Inc.; Richmond, Calif.) or vacuum liquid chromatography onsilica gel. The calanolides are then purified from those columnfractions that demonstrate HIV-inhibitory activity by sequential HPLC onsilica (elution with 3:7 EtOAc-hexane) and C₁₈ reversed-phase (elutionwith 9:1 MeOH-H₂ O) columns. Using this general procedure, eithercalanolide A or calanolide B, or both, can be obtained, for example, inan overall yield of about 0.05-0.2%; related compounds also present inthe extract can be obtained in similar yields. The isolation ofcalanolides and derivatives thereof from C. lanigerum var.austrocoriaceum is described in greater detail in Example 2.

Antiviral calanolides and related antiviral compounds from latex ofCalophyllum plants. A simplified procedure can be used to obtain highyields of antiviral calanolides or related antiviral compoundsnon-destructively from Calophyllum plants. For example, 10-100 grams ofcrude Calophyllum plant latex, which can be harvested from Calophyllumplants as described further in Example 7 below, is extracted with CHCl₃:MeOH (1:1). The solution is filtered and evaporated to yield a residuetypically amounting to 60-70% of the mass of the original latex. Theantiviral compounds may then be separated and purified in high yields,typically 10-40% of the total residue mass, by HPLC on silica elutedwith hexane:EtOAc (7:3). The isolation of costatolide (compound 9 ofFIG. 2) and soulattrolide (compound 14 of FIG. 2) from latex ofCalophyllum teysmannii var. inophylloide is described more specificallyin Example 8 herein.

Rationale and collection of latex from Calophyllum plants. In theprocess of plant drug discovery and development, after an initial small(0.5-1 kg dry weight screening-sized) sample shows biological activityof interest, and the promising active compound(s) is (are) identified, alarger quantity (10-20 kg dry weight) of material typically must berecollected, in order to permit reisolation of a larger quantity of thepure compound(s) for further preclinical investigation. If the resultsof the preclinical studies continue to be promising, an even largerquantity of plant material (50-1000 kg dry weight) of that species willneed to be recollected for further developmental studies. Very muchlarger amounts will be required for clinical use. Collection of leaf,twig, stembark, and root samples in large quantities is destructive andmay be detrimental to the plant species in question and to the forestenvironment.

Considering that Calophyllum produces latex, if the latex could be shownto possess the same biological activity, i.e., containing the sameanti-HIV compound(s) of interest, it would be most desirable to use thelatex as a source (raw) material for re-isolation work and, eventually,for industrial production of the drug. Latex is exuded when a treebarkis slashed (cut), and the resinous exudate may be collectedperiodically, without damage to the tree itself. Examples of suchnon-destructive harvest methods are provided by the para rubber tree(Hevea brasiliensis) and the sugar maple tree (Acer saccharum). Such amethod is now clearly applicable to the harvest of Calophyllum latex,which can lead to the sustainable or continued utilization of the forestresource.

Latex collections of Calophyllum were initiated on Jul. 19, 1992, inSarawak; then on Oct. 9, 1992, in the Singapore Garden jungle forCalophyllum lanigerum var. austrocoriaceum; and on Oct. 11-13, 1992,again in Sarawak, for various Calophyllum species, including Calophyllumlanigerum var. austrocoriaceum. Finally, on Jan. 7-8, 1993, fieldexperiments were run to collect latex samples from trees of C.teysmannii var. inophylloide in the Sampedi Forest Reserve. An adequatelatex yield was obtained from 5 trees, by collecting latex samples fromeach of these trees on 3 different occasions during a total period of 2days (Jan. 7-8, 1993). A fourth collection from the same trees was doneon the following week of January 11, 1993. Approximately 100 grams oflatex was initially obtained. Example 7 below sets forth in furtherdetail the non-destructive harvest of latex from Calophyllum tyesmanniivar. inophylloide; Example 8 illustrates the isolation and purificationof calanolide-related antiviral compounds, costatolide andsoulattrolide, from such latex. Based on the fact that latex scrapedfrom 6-month old wounds that had healed still showed the presence ofcostatolide on the TLC plates, and the fact that 3 scraping operationson the same slashes on a tree could be made in different occasions, eachof which yielded an appreciable latex quantity, it is apparent thatlatex harvest is a sustainable harvest method.

Determination Of Chemical Structures Of Antiviral Calanolides AndRelated Antiviral Compounds From Calophyllum Plants

Chemical structures of calanolides and related compounds and derivativesthereof which can be isolated by the above general procedure fromextracts from Calophyllum plants are shown in FIG. 1. The proofs of thestructures can be obtained by a combination of methods, includingprimary spectral analyses (e.g., high-resolution NMR and massspectrometry, infrared and UV spectroscopy), comparisons of spectral andphysicochemical properties with related literature precedents, and byselected derivatization procedures, such as for determination ofabsolute stereochemistry. The structure proofs for calanolides A, B, andderivatives thereof from C. lanigerum var. austrocoriaceum are describedin detail in Example 3 and summarized in Tables 1 and 2.

It will be appreciated by one who is skilled in the art that antiviralcalanolides, related antiviral compounds, and derivatives thereof, otherthan those specifically described herein, may be isolated from otherCalophyllum species and other natural sources and that such antiviralcalanolides, related antiviral compounds, and derivatives thereof alsomay be synthesized chemically. Generic structural series, designated as"Series 1" and "Series 2," are described in detail in Example 5 andFIGS. 5A and 5B, respectively. Members of series 1 can be converted tothe corresponding 7,8-dihydro compounds of series 2, by catalytichydrogenation. Example 9 below more specifically illustrates thepreparation of antiviral compounds 7,8-dihydrocalanolide A,7,8-dihydrocalanolide B, 7,8-dihydrocostatolide, and7,8-dihydrosoulattrolide; the structures of these compounds are shown inFIGS. 6 A-D.

Antiviral Activity

The antiviral activity of calanolides and related compounds andderivatives thereof can be demonstrated further in a series ofinterrelated in vitro assays (Gulakowski, R. J., et al., J. Virol.Methods, 33, 87-100 (1991)), which accurately predict antiviral activityof chemical compounds in humans. These assays measure the ability ofcompounds to prevent the replication of HIV and/or the cytopathiceffects of HIV on human target cells. These measurements directlycorrelate with the pathogenesis of HIV-induced disease in vivo.Accordingly, the results of the analysis of the antiviral activity ofcalanolides and related compounds and derivatives thereof from C.lanigerum, as set forth in Example 3 and as illustrated in FIGS. 4 A-Dand FIGS. 7 A-H, are believed to predict accurately the antiviralactivity of these compounds in humans.

The compounds which are the subject of the present invention can beshown to inhibit a retrovirus, such as the human immunodeficiency virus,specifically HIV-1 and HIV-2. As one skilled in the art will appreciate,the compounds of the present invention probably will inhibit otherretroviruses and may inhibit viruses, other than retroviruses. Examplesof viruses that may be treated in accordance with the present inventioninclude, but are not limited to, Type C and Type D retroviruses, HTLV-1,HTLV-2, HIV, FLV, SIV, MLV, BLV, BIV, equine infectious, anemia virus,avian sarcoma viruses, such as rous sarcoma virus (RSV), hepatitis typeA, B, non-A, and non-B viruses, herpes viruses, cytomegaloviruses,influenza viruses, arboviruses, varicella viruses, measles, mumps andrubella viruses.

Compositions And Formulations

The calanolides and related compounds and derivatives thereof may beformulated into various compositions for use in therapeutic andprophylactic treatment methods. The present inventive composition may beused to treat a virally infected animal, such as a human. Thecompositions of the present invention are particularly useful ininhibiting the growth or replication of a virus, such as a retrovirus,in particular a human immunodeficiency virus, specifically HIV-1 andHIV-2. The compositions also are expected to be useful in the treatmentof animals, such as humans, infected with other viruses, such as thoselisted above. Furthermore, such compositions should find utility in theprophylactic treatment of animals, such as humans, who are at risk forviral infection.

Compositions for use in the prophylactic or therapeutic treatmentmethods of the present invention comprise one or more calanolides orrelated compounds and derivatives thereof and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers are well-knownto those who are skilled in the art, as are suitable methods ofadministration. The choice of carrier will be determined in part by theparticular calanolide compound, as well as by the particular method usedto administer the composition.

One skilled in the art will appreciate that various routes ofadministering a drug are available and, although more than one route maybe used to administer a particular drug, a particular route may providea more immediate and more effective reaction than another route.Furthermore, one skilled in the art will appreciate that the particularpharmaceutical carrier employed will depend, in part, upon theparticular compound employed and the chosen route of administration.Accordingly, there is a wide variety of suitable formulations of thecomposition of the present invention.

Formulations suitable for oral administration may consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solids or granules; solutions or suspensions in anaqueous liquid; and oil-in-water emulsions or water-in-oil emulsions.Tablet forms may include one or more of lactose, mannitol, corn starch,potato starch, microcrystalline cellulose, acacia, gelatin, colloidalsilicon dioxide, croscarmellose sodium, talc, magnesium stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers.

The calanolides and related compounds and derivatives thereof, alone orin combination with other antiviral compounds, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, gels, and the like containing, inaddition to the active ingredient, such carriers as are known in theart.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate. Similarly, the active ingredient maybe combined with a lubricant as a coating on a condom.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which may containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that may include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations may be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a prophylacticor therapeutic response in the infected individual over a reasonabletime frame. The dose will be determined by the strength of theparticular antiviral compound employed, the severity of the diseasestate, as well as the body weight and age of the infected individual.The size of the dose also will be determined by the existence of anyadverse side effects that may accompany the particular compoundemployed. It is always desirable, whenever possible, to keep adverseside effects to a minimum.

The dosage may be in unit dosage form, such as a tablet or capsule. Theterm "unit dosage form" as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a calanolide or relatedcompound or derivative thereof, alone or in combination with otherantiviral agents, calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier, or vehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular compound or compounds employed and the effectto be achieved, as well as the pharmacodynamics associated with eachcompound in the host. The dose administered should be an "antiviraleffective amount" or an amount necessary to achieve an "effective level"in the individual patient.

Antivral Therapy

Since the "effective level" is used as the preferred endpoint fordosing, the actual dose and schedule may vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The "effective level" may be defined, for example, as theblood or tissue level desired in the patient that corresponds to aconcentration of one or more calanolides, or related compounds orderivatives thereof, which inhibits a virus such as HIV in an assayknown to predict for clinical antiviral activity of chemical compounds.The "effective level" for compounds which are the subject of the presentinvention also may vary when the compositions of the present inventionare used in combination with AZT or other known antiviral compounds orcombinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired "effectiveconcentration" in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the "effectiveconcentration" of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it may bedesirable to utilize a "mega-dosing" regimen, wherein a large dose ofthe calanolide or derivative thereof is administered, time is allowedfor the compound to act, and then a suitable reagent is administered tothe individual to inactivate the calanolide or derivative thereof.

The pharmaceutical composition may contain other pharmaceuticals, inconjunction with the calanolide or related compound or derivativethereof, when used to therapeutically treat acquired immunodeficiencysyndrome (AIDS). Representative examples of these additionalpharmaceuticals include antiviral compounds, immunomodulators,immunostimulants, and antibiotics. Exemplary antiviral compounds include3'-azido-2',3'-dideoxythymidine (AZT), 2'3'-dideoxyinosine (ddI),2'3'-dideoxycytidine (ddC), 2'3'-didehydro-2'3'-dideoxythymidine (D4T),9-(1,3-dihydroxy-2-propoxymethyl)guanine (gancyclovir), fluorinateddideoxynucleotides such as 3'-fluoro-2'3-dideoxythymidine, nonnucleosidecompounds such as6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e][1,4]diazepin-6-one(nevirapine) (Shih et al., PNAS, 88, 9878-9882 (1991)), TIBO and analogsand derivatives such as(+)-S,4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione(R82913) (White et al., Antiviral Research, 16, 257-266 (1991)), Ro31-8959 (Craig et al., Antiviral Research, 16, 295-305 (1991)), BI-RJ-70(Shih et al., supra), 9-(2-hydroxyethoxy-methyl)guanine (acyclovir),α-interferon, recombinant CD4 (Merigan et al., The American Journal ofMedicine, 90 (Suppl. 4A), 8S-17S (1991)), pyridine analogs such as(3-[(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one(L-696,229), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine (HEPT),carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir), and[2',5'-Bis-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine(TSAO-T). Exempletive immunomodulators and immunostimulants includevarious interleukins, CD4, cytokines, antibody preparations, bloodtransfusions, and cell transfusions. Exempletive antibiotics includeantifungal agents, antibacterial agents, and anti-Pneumocystis cariniiagents.

Administration of the inhibitory compound with other anti-retroviralagents and particularly with known reverse transcriptase (RT)inhibitors, such as ddC, AZT, ddI, ddA, or other inhibitors that actagainst other HIV proteins, such as anti-TAT agents, will generallyinhibit most or all replicative stages of the viral life cycle. Thedosages of ddC and AZT used in AIDS or ARC patients have been published.A virustatic range of ddC is generally between 0.05 μM to 1.0 μM. Arange of about 0.005-0.25 mg/kg body weight is virustatic in mostpatients. The preliminary dose ranges for oral administration aresomewhat broader, for example 0.001 to 0.25 mg/kg given in one or moredoses at intervals of 2, 4, 6, 8, 12, etc. hours. Currently 0.01 mg/kgbody weight ddC given every 8 hours is preferred. When given in combinedtherapy, the other antiviral compound, for example, may be given at thesame time as the calanolide compound or the dosing may be staggered asdesired. The two drugs also may be combined in a composition. Doses ofeach may be less when used in combination than when either is usedalone.

EXAMPLES

The present inventive compounds and methods are further described in thecontext of the following examples. These examples serve to illustratefurther the present invention and are not intended to limit the scope ofthe invention.

Example 1

This example sets forth more specifically the taxonomic characteristicsof the Calophyllum lanigerum var. austrocoriaceum specimen (Burley & Lee351) used in example 2: tree about 8 m tall, dbh (diameter at breasthigh) 15 cm; bark light brownish with dark brown longitudinal rows oflenticels, inner bark red, underbark orange; twigs somewhat flattenedand more-or-less angled, dark brown and covered with short trichomes ofthe same color, terminal buds plump, 1.5-2 cm long, thickish, beset withrusty brown, soft-tomentose pubescence; leaves coriaceous, bladesnarrowly ovate to oblong, 7-17 cm long, 3-5 cm wide, chestnut brown indry state, latex canals as prominent as veins, about 10 veins per 5 mm,petiole 1-1.5 cm long; inflorescence few-flowered (3-5 flowers perinflorescence), axis 3-3.5 cm long, lowest internode 0.5-1 cm long;fruits young, globose to ellipsoid, 0.5-1 cm across, green in freshcondition, dark chestnut brown when dry.

Example 2

This example illustrates the isolation of calanolides and derivativesthereof from extracts of Calophyllum lanigerum var. austrocoriaceum.Dried fruit and twigs (763 g) of Calophyllum lanigerum var.austrocoriaceum were stored at -20° until the material was ground,percolated in 1:1 CH₂ Cl₂ /MeOH, and washed with 100% MeOH. Removal ofthe solvent under reduced pressure provided 72.5 g of organic extract.

A 10 g portion of the organic extract was subjected to a solvent-solventpartitioning protocol. A 10 g portion of the organic extract wassuspended in 500 ml CH₃ OH--H₂ O (9:1) and extracted with hexane (3×300ml). The water content of the polar phase was increased to 20%,whereupon a second extraction was conducted with CCl₄ (3×300 ml). Theanti-HIV activity of all fractions was assessed using a routine primaryscreening assay (Weislow, O. S., et al., J. Natl. Cancer Inst., 81(8),577-586 (1989)) at each chromatographic step. Anti-HIV activity wasconcentrated in the hexane (770 mg) and CCl₄ (590 mg) soluble fractions.The active fractions were individually separated by vacuum liquidchromatography (VLC) on 10 g of silica gel using mixtures ofhexane/EtOAc. The active constituents that eluted with 10-25% EtOAc werecombined, based upon TLC and ¹ H NMR profiles, to provide two activefractions. The individual fractions were further separated by VLC onsilica, using gradual step-gradient elution with hexane/EtOAc mixtures.Final purification gave eight compounds (1-8), the structures of whichare given in FIG. 1. Silica HPLC eluted with 7:3 hexane/EtOAc gavecalanolide A (1) (11.7 mg), calanolide B (4) (5.0 mg), and compound 7(12.5 mg). Purification of the other components was effected by C₁₈ HPLCwith 9:1 MeOH/H₂ O to give 12-acetoxycalanolide A (2) (7 mg),12-methoxycalanolide A (3) (5 mg), 12-methoxycalanolide B (5) (16 mg),compound 6 (4 mg), and compound 8 (11 mg). FIG. 2 shows six relatedstructures (9-14) previously reported from other sources. By comparingthe structures and the spectral and physicochemical properties of thenovel compounds of FIG. 1 with those of the known compounds of FIG. 2,it can be seen that the compounds 1-8 of the present invention differfrom those compounds previously reported from other sources.

Example 3

This example sets out the structure proofs for calanolides A and B andrelated derivatives from Calophyllum lanigerum var. austrocoriaceum,which were obtained in Example 1. Calanolide A (1) was isolated as anoptically active oil, [α]_(D) =+60°, which gave a HREIMS parent ion atm/z 370.1764 daltons, indicating a molecular formula of C₂₂ H₂₆ O₅. Themass spectrum contained significant fragment ions for M⁺ --CH₃ (m/z 355,100%), M⁺ --CH₃ --H₂ O (m/z 337, 12%) and M⁺ --C₅ H₁₁ (m/z 299, 29%).The infrared spectrum showed bands corresponding to hydroxyl (3300 cm⁻¹)and carbonyl (1735 cm⁻¹) groups. Resonances for 11 sp² carbons in the ¹³C NMR spectrum revealed a conjugated ester (δ 160.4), a disubstitutedolefin conjugated to a phenyl group (δ 126.9 [1H] and 116.5 [1H]), atrisubstituted olefin conjugated to a carbonyl (δ 158.9 and 110.1 [1H]),and a fully substituted benzene ring bearing three oxygen moieties (δ154.5, 153.1, 151.1, 106.3, 106.4, and 104.0). Taking into account thenumber of double bond equivalents implicit in the molecular formula,calanolide A (1) was determined to be tetracyclic. The ¹ H NMR spectrumshowed two methyl singlets (δ 1.49 and 1.44), two methyl doublets (δ1.44 and 1.13), and a methyl triplet (δ 1.01). Additional proton signalsincluded those of an allylic methylene (δ 2.87 [2H], m), an aliphaticmethylene (1.63 [2H], m), and three olefin protons (δ 6.60 d, J=9.5 Hz;5.92 t, J=1.0 Hz; 5.52 d, J=9.5 Hz). These data suggested thatcalanolide A (1) was a coumarin derivative related to costatolide (9), ametabolite previously reported from Calophyllum costatum (Stout, G. H.,J. Org. Chem., 29, 3604-3609 (1964)) .

One-bond and long-range proton detected heteronuclear correlationexperiments (HMQC and HMBC) allowed the complete assignment of both the¹ H NMR (Table 1) and ¹³ C NMR spectra (Table 2) of calanolide A (1).Key correlations included those between H8 and carbons 4b, 6, 8a, and8b, which helped to establish the position of the 2,2-dimethylchromenesystem. Placement of the n-propyl group at C4 was aided by a 1.0 Hzallylic coupling between the C13 allylic methylene protons and the C3olefin proton, and by three-bond heteronuclear correlations from the C13methylene protons to C3 and C4a. The remaining substitution patternabout the coumarin nucleus was defined by correlations between H12 andcarbons 8b, 10, 11, 12a, 12b, and 19. This confirmed that, althoughcalanolide A (1) had the same skeleton as costatolide (9), they differedin the relative stereochemistry of their substituents about the2,3-dimethylchromanol ring.

In the ¹ H NMR spectrum of compound 1, the H12 benzylic carbinol protonshowed a 8.0 Hz coupling to H11, which revealed that these two protonshad a trans-diaxial orientation. A 9.0 Hz coupling between H11 and H10established that H10 also was axial. This assignment was supported bynOe enhancements (3%) observed between the diaxial H10 and H12 protons.Calanolide A (1) was thus determined to be a diastereomer of costatolide(9), which showed J₁₀₋₁₁ and J₁₁₋₁₂ of 10.5 Hz and 3.5 Hz, respectively(Stout, G. H., et al., J. Org. Chem., 29, 3604-3609 (1964)). Two relatedcoumarin derivatives, inophyllum B (10) (Bandara, B. M. R., et al.,Phytochemistry, 25(2), 425-428 (1986)) and cordatolide A (11)(Dharmaratne, H. R. W., et al., Phytochemistry, 24, 1553-1557 (1985))reportedly have the same relative stereochemical features about thechromanol ring as those found in compound 1, but differ in their C4substituents. The J₁₀₋₁₁ and J₁₁₋₁₂ coupling constants observed incalanolide A (1) agree closely with those described in the above-citedpublications for compounds 10 and 11.

Other physicochemical and spectral data for calanolide A (1) were asfollows, [α]_(D) +60° (CHCl₃, c 0.7); UV λ_(max) (MeOH) 325 (ε 13,700),284 (ε 22,800), 228 (ε 22,200) nm; IR (film) ν_(max) 3300, 2966, 1735,1713, 1583, 1111 cm⁻¹ ; HREIMS obs. m/z 370.1764, calc'd for C₂₂ H₂₆ O₅,370.1780; low res. MS m/z 370 (38%), 355 (100%), 337 (12%), 299 (29%).

12-Acetoxycalanolide A (2), [α]_(D) =+20°, gave a parent ion by HREIMSat m/z 412.1825 daltons corresponding to a molecular formula of C₂₄ H₂₈O₆. Significant fragment ions were observed for M⁺ --CH₃ (m/z 397, 41%)M⁺ --AcOH (m/z 352 30%) and M⁺ --AcOH--CH₃ (m/z 337, 100%). The presenceof an acetate group was suggested by a sharp 3H singlet in the ¹ H NMRspectrum at δ 2.10 and ¹³ C NMR resonances at δ 21.2 (3H) and 170.7. Theremaining ¹ H and ¹³ C NMR signals for compound 2 were very similar tothose recorded for calanolide A (1), except that the H12 resonance incompound 2 was shifted downfield to δ 5.97. This suggested that compoundZ was the 12-acetoxy derivative of calanolide A (1). The J₁₀₋₁₁ (6.5 Hz)and J₁₁₋₁₂ (6.0 Hz) couplings in compound 2 supported a pseudoaxialorientation of the chromanol ring protons. The slightly diminishedchromanol proton couplings in compound 2 conceivably resulted from aslight twisting of the flexible chromanol ring. Further evidence for theproposed substituent configuration was provided by difference nOeenhancements of 2% measured between H10 and H12.

Other physicochemical and spectral data for compound 2 were as follows:[α]_(D) +20° (CHCl₃, c 0.5); IR (film) ν_(max) 2960, 1738, 1585, 1376,1230, 1138 cm⁻¹ ; HREIMS obs. m/z 412.1825, calc'd for C₂₄ H₂₈ O₆,412.1886; low res. MS m/z 412 (13%), 397 (41%), 352 (30%), 337 (100%),299 (8%).

The HREIMS of 12-methoxycalanolide A (3), [α]_(D) =+32°, showed a parention at m/z 384.1924 daltons, corresponding to a molecular formula of C₂₃H₂₈ O₅. Significant fragment ions observed for M⁺ --CH₃ (m/z 369, 12%),M⁺ --CH₃ OH (m/z 352, 9%) and M⁺ --CH₃ OH--CH₃ (m/z 337, 100%) suggestedthe presence of a methoxyl group, which was confirmed by a ¹ H NMRsinglet (δ 3.59, 3H) and a corresponding carbon resonance at δ 57.6. The¹ H and ¹³ C NMR spectra revealed that compound 3 had the same skeletonas calanolide A (1). However, important differences were observed in thesignals for some of the chromanol ring substituents. In addition to thevicinal couplings of J₁₀₋₁₁ (3.5 Hz) and J₁₁₋₁₂ (3.7 Hz), a W couplingof 1.3 Hz was observed between H10 and H12 in compound 3. The W couplingrequired a pseudodiequatorial configuration for the C10 and C12 protons.Significant nOe enhancements between H11 and both the C10 methyl group(3.5%) and the C12 methoxyl group (3.5%) indicated that H11 was cis tothese two substituents and, therefore, had an equatorial orientationabout the chromanol ring. It appeared that, in 12-methoxycalanolide A(3), the preferred conformation of the chromanol ring was invertedrelative to calanolide A (1). Thus, while H10, H11, and H12 wereoriented α, β, α respectively in both compounds, in calanolide A (1) allthree protons were axial, and in 12-methoxycalanolide A (3) they wereall equatorial.

Other physicochemical and spectral data for compound 3 were as follows:[α]_(D) +32° (CHCl₃, c 0.8); IR (film) ν_(max) 2960, 1731, 1584, 1380,1137, cm⁻¹ ; HREIMS obs. m/z 384.1924, calc'd for C₂₃ H₂₈ O₅, 384.1937;low res. MS m/z 384 (5%), 369 (12%), 352 (9%), 337 (100%).

Calanolide B (4), [α]_(D) =+8°, was isomeric to calanolide A (1), as italso showed a HREIMS parent ion at m/z 370.1747 daltons, correspondingto C₂₂ H₂₆ O₅. The ¹ H and ¹³ C NMR spectra of calanolide B (4) werevirtually identical to those from calanolide A (1), with the exceptionof some variations in signals from the chromanol ring. It was clear fromthe spectral data that compound 4 differed from compound 1 only in thestereochemical disposition of the chromanol ring substituents.Proton-proton coupling constant analysis showed a 10.5 Hz J₁₀₋₁₁coupling and a 3.3 Hz J₁₁₋₁₂ coupling. Thus, H10 and H11 weretrans-diaxial while H11 and H12 were in a cis configuration with H12 ina pseudoequatorial orientation. Calanolide B (4) had the same relativestereochemistry as costatolide (9) but its optical rotation was oppositein sign to that reported for compound 9 (Stout, G. H., et al., J. Org.Chem., 29, 3604-3609 (1964)); therefore, compounds 4 and 9 areenantiomeric.

Other physicochemical and spectral data for calanolide B (4) were asfollows: [α]_(D) +10° (acetone, c 1.0) UV ν_(max) (MeOH) 325 (ε 13,700),284 (ε 22,800), 228 (ε 22,200) nm; IR (film) ν_(max) 3470, 2970, 1732,1587 cm⁻¹ ; HREIMS obs. m/z 370.1747, calc'd for C₂₂ H₂₆ O₅, 370.1780;low res. MS m/z 370 (3%), 355 (100%), 337 (13%), 300 (5%), 299 (20%).

12-Methoxycalanolide B (5), [α]_(D) =+34°, provided a HREIMS parent ionat m/z 384.1890 daltons appropriate for a molecular formula of C₂₃ H₂₈O₅. Additional fragment ions were seen for M⁺ --CH₃ (m/z 369, 12 %), M⁺--CH₃ OH (m/z 352, 13%) and M⁺ --CH₃ OH--CH₃ (m/z 337, 100%). The ¹ Hand ¹³ C NMR spectra of compound 5 were virtually identical to thoserecorded for compound 4, with the addition of a sharp 3H singlet at δ3.58 and a corresponding carbon resonance at δ 59.4. These dataindicated that compound 5 was the 12-methoxyl derivative of calanolide B(4). This assignment was confirmed by acid hydrolysis of 2 mg ofcompound 5 in 400 μl of THF/H₂ O and 8 μl of 6N HCl at room temperaturefor 48 hr to provide compound 4 as the only product.

Other physicochemical and spectral data for compound 5 were as follows:[α]_(D) +34° (CHCl₃, c 0.5); IR (film) ν_(max) 2966, 1734, 1716, 1700,1558, 1540, 1506, 1457 cm⁻¹ ; HREIMS obs. m/z 384.1890, calc'd for C₂₃H₂₈ O₅, 384.1937; low res. MS m/z 384 (4%), 369 (12%), 352 (13%), 337(100%).

Compound 6, [α]_(D) =+68°, also was isomeric with calanolide A (1),since it showed similarly a HREIMS parent ion at m/z 370.1695 daltons,consistent with a molecular formula of C₂₂ H₂₆ O₅. Fragment ions werefound at M⁺ --CH₃ (m/z 355, 100%), M⁺ --CH₃ --H₂ O (m/z 337, 25%) and M⁺--C₅ H₁₁ (m/z 299, 35%) Again the only notable differences between the ¹H and ¹³ C NMR spectra of 6 and those recorded for compound 1 were theresonances associated with the chromanol ring. The J₁₀₋₁₁ in compound 6was 2.5 Hz, while J₁₁₋₁₂ was 6.0 Hz. These coupling constants wereinsufficient to define the relative stereochemistry of carbons 10, 11,and 12. However, the C12 hydroxyl proton gave a sharpened peak with a1.5 Hz coupling to H12 which suggested that the rate of exchange of theOH proton was reduced due to hydrogen bonding to O1. Hydrogen bonding toO1 would require an equatorial OH at C12. A 5% nOe enhancement betweenH10 and H12 confirmed that these protons each had axial orientations.Therefore, H11 had to be equatorial. Compound 6 was thus the C11 epimerof calanolide A (1) and had the same substitution pattern and relativestereochemistry about the chromanol ring as the previously describedcoumarin derivative inophyllum A (12) (Bandara, B. M. R., et al.,Phytochemistry, 25, 425-428 (1986)); the J₁₀₋₁₁ (3.3 Hz) and J₁₁₋₁₂values (5.4 Hz) reported earlier for compound 12 were in good agreementwith the respective couplings observed in compound 6.

Other physicochemical and spectral data for compound 6 were as follows:[α]_(D) +68° (CHCl₃, c 0.7); IR (film) ν_(max) 2960, 1729, 1620, 1582,1120 cm⁻¹ ; HREIMS obs. m/z 370.1695, calc'd for C22H26O₅, 370.1780; lowres. MS m/z 370 (52%), 355 (100%), 337 (25%), 200 (35%).

Compound 7, [α]_(D) =+60°, provided a HREIMS molecular ion at m/z368.1213 appropriate for a molecular formula of C₂₂ H₂₄ O₅. Thisrequired that compound 7 had one additional unsaturation equivalentrelative to calanolide A (1). The infrared spectrum, with bands at 1734and 1697 cm⁻¹ suggested the presence of an additional carbonyl group.Heteronuclear correlation experiments allowed the complete assignment ofthe ¹ H and ¹³ C NMR spectra of compound 7. While the ¹³ C NMR spectrumof compound 7 was quite similar to that of calanolide A (1), the C12peak in compound 7 was shifted downfield to δ 192.9, indicative of anα,β-unsaturated ketone functionality. A shift of the C11 protonresonance in compound 7 to δ 2.61 supported its placement a to a ketonecarbonyl.

The small coupling measured between H10 and H11 (J₁₀₋₁₁ =3.0 Hz)indicated that at least one of these protons was equatorial. Thepreviously described oxidation product (13) of soulattrolide (14)(Gunasekera, S. P., et al., J. Chem. Soc. Perkin I, 1505-1511 (1977))contains a similar 2,3-dimethyl-benzopyranone ring system. In compound13, H10 and H11 are trans, and a J₁₀₋₁₁ coupling of 11.0 Hz wasreported. This indicated that when the H10 and H11 protons were trans,the ring adopted a conformation with these two protons in a diaxialorientation. Therefore, the relative stereochemistry of the H10 and H11protons in compound 7 had to be cis. The absolute stereochemistry at C10and C11 has not been determined and, therefore, both of thecorresponding methyls have been drawn arbitrarily as α in FIG. 1.

Other physicochemical and spectral data for compound 7 were as follows:[α]_(D) +60° (CHCl₃, c 0.5); IR (film) ν_(max) 2960, 1734, 1697, 1684,1575, 1558 cm⁻¹ ; HREIMS obs. m/z 368.1213, calc'd for C₂₂ H₂₄ O₅,368.1624; low res. MS m/z 368 (25%), 353 (100%), 297 (68%).

Compound 8, [α]_(D) =+30° had a molecular formula of C₂₂ H₂₈ O₆, asindicated by the HREIMS parent ion at m/z 388.1890 daltons Fragment ionsappropriate for M⁺ --CH₃ (m/z 373, 100%), M⁺ --C₃ H₇ (m/z 345, 3%), M⁺--CO₂ CH₃ (m/z 329, 5%), M⁺ --C₃ H₇ O₂ (m/z 313, 3%) and M⁺ --COCHCH₃CHOHCH₃ (m/z 287, 3%) were also observed. The complete ¹ H and ¹³ C NMRspectra of compound 8 were assigned with information provided from nOeexperiments and heteronuclear correlations. The ¹³ C NMR spectrumcontained signals for an unsaturated ketone (δ 201.0), a saturated ester(δ 178.6), a disubstituted olefin (δ 125.6 [1H] and 115.6 [1H]) and afully substituted benzene ring bearing three oxygens (δ 160.0 [2C],157.3, 108.9, 102.6 and 101.2). Therefore, compound 8 had only three ofthe four rings found in the other members of the calanolide series. Incontrast to compounds 1-7, which gave vivid blue spots on TLC whencharred with vanillin/H₂ SO₄, compound 8 gave a brownish-green spot.

The ¹ H and ¹³ C NMR spectra of compound 8 showed some resonances thatcorresponded closely to the coumarin and 2,2-dimethylchromene ringsystems of compounds 1-7. However, the C3/C4 double bond in compounds1-7 was fully saturated in compound 8. For clarity of discussion, asdepicted in FIG. 2, the carbon atoms have been numbered in compound 8 ina scheme that is analogous to the numbering shown for calanolide A (1)and which likewise applies to the others of the series, i.e., compounds2-7. The saturation of the double bond resulted in a methylene (δ 2.81dd, J=15.0, 9.0 Hz and 2.67 dd, J=15.0, 6.5 Hz) α to the lactonecarbonyl that was coupled to the C4 benzylic methine (δ 3.67 m). The C4proton also showed heteronuclear correlations to C2, C3, C4a, C12b, C13and C14, which supported the presence of a 3,4-dihydrocoumarin skeletonwith an n-propyl substituent at C4. Heteronuclear correlations,including those between H8 and C4b, C6, and C8b, confirmed the placementof the chromene functionality on the coumarin ring system. Thissuggested that the chromanol ring system present in compounds 1∂14 wasopen in compound 8.

The position of the phenol on C8b was established by heteronuclearcorrelations from the phenolic proton to C8a, C8b, and C12a and an nOeinteraction with H8. Since the remaining ketone group in the moleculewas unsaturated, it had to be located on C12. In this position it couldaccept a hydrogen bond from the C8b phenol proton, which appeared as asharp singlet at δ 12.40. The downfield shift of H11 (δ 2.52 dq, J=3.5,6.5 Hz) was appropriate for a methine located α to a ketone. The C10carbinol methine proton (δ 4.49 dq, J=7.0, 3.5 Hz) showed vicinalcoupling to H11 and to the C18 methyl group. All heteronuclearcorrelations, including those from the C19 methyl protons to C10, C11and C12, were fully consistent with the proposed structure for compound8.

Other physicochemical and spectral data for compound 8 (FIG. 1) were asfollows: [α]_(D) +30° (CHCl₃, c 0.5); IR (film) ν_(max) 2960, 1706,1644, 1625, 1442, 1131 cm⁻¹ ; HREIMS obs. m/z 388.1890, calc'd for C₂₂H₂₈ O₆, 388.1886; low res. MS m/z 388 (19%), 373 (100%), 345 (3%), 329(5%), 313 (3%), 287 (3%).

The absolute stereochemistry of calanolide A (1) and calanolide B (4)was determined by a modified Mosher's method (Ohtani, I., et al.,Tetrahedron Lett., 30(24), 3147-3150 (1989); J. Org. Chem., 56,1296-1298 (1991)). The technique utilized anisotropic shifts induced inthe ¹ H NMR spectra of methoxy(trifluoromethyl)-phenylacetic (MTPA)esters of secondary alcohols to define the absolute stereochemistry.Both (+)-(R)- and (-)-(S)-MTPA esters (FIG. 3) of compounds 1 and 4 wereprepared.

A solution of (R)-α-methoxy-α-(trifluoromethyl)phenylacetic acidchloride ((R)-MTPA chloride) (2.5 mg in 50 μl of benzene) was added to 3mg of calanolide A (1) dissolved in 3 ml of dry benzene. A 0.03 mgaliquot of dimethylaminopyridine and 10 μl of triethylamine were added,and the reaction mixture was refluxed. After 3 h, a second 2.5 mgportion of (R)-MTPA chloride was added, and the reaction was refluxedfor an additional 21 h. When the mixture was cooled, 10 ml of benzenewere added and the organic phase was successively washed with 10% HCl, NNaHCO₃, and H₂ O. The solution was dried over Na₂ SO₄, evaporated todryness, and then quickly chromatographed on a short plug (1×2 cm) ofsilica, eluting with mixtures of hexane/EtOAc. A compound which appearedto be an elimination product eluted first with 5% EtOAc, while thedesired (S)-MTPA ester eluted with 12% EtOAc. The same procedure wasrepeated with (S)-MTPA chloride to give the (R)-MTPA ester. The (S)- and(R)-MTPA esters of calanolide B (4) were prepared in an identicalfashion, with the exception that, after the second addition of the MTPAchloride, the reaction mixture was refluxed for only an additional 2 h(total time of reflux=5 h). The Δδ values (FIG. 3) from the 500 MHz ¹ HNMR spectra were calculated (Δδ=δ_(S) -δ_(R)). By this method, theabsolute configuration of C12 was determined to be 12S in calanolide A(1) and 12R in calanolide B (4). As established earlier, calanolide A(1) [10R, 11R, 12S] and calanolide B (4) [10R, 11R, 12R] were C12epimers.

Esterification of calanolide A (1) occurred slowly (24 hr reflux forcompound 1 vs. 5 hr for compound 4), and, by ¹ H NMR analysis,esterification appeared to cause a change in the conformation of thechromanol ring. The methyls and the ester group flipped from equatorialto axial positions in the MTPA ester of compound 1, as J₁₀₋₁₁ =2.5 Hz(previously 9.0 Hz) and J₁₁₋₁₂ =2.5 Hz (previously 8.0 Hz). In addition,a 4-bond W coupling of 1.5 Hz between H10 and H12 also could beobserved. Similar changes in the conformation of the chromanol ring werepreviously noted for compound 3. The anisotropic shifts induced in theMTPA esters indicated that the bulky MTPA group was sterically repulsedby the coumarin ring lactone. Thus, the plane that divided themolecule's proton resonances into Δδ-positive and Δδ-negative did notcleanly bisect the dihydropyran ring through C12 and O9. In calanolide A(1), the dividing plane seemed to come closer to C6 and, in calanolide B(4), to C8b. Due to the 1,3-diaxial orientation of the C10β-methyl andthe C12β-ester group in the MTPA ester of compound 1, the former methylgroup was very strongly influenced by the ester (Δδ=-240 in comparisonto -16 measured for the C11α-methyl).

Summaries of the 500 MHz ¹ H-NMR data and the 125 MHz ¹³ C-NMR data forcompounds 1-8 (FIG. 1) are provided in Tables 1 and 2, respectively.

                                      TABLE 1                                     __________________________________________________________________________    500 MHz .sup.1 H NMR Data for Compounds 1-8.sup.a                             Proton                                                                        #   1      2      3      4      5      6       7       8                      __________________________________________________________________________     3  5.92,t,                                                                              5.93,t,                                                                              5.94,t,                                                                              5.93,t,                                                                              5.92,t,                                                                              5.94,t, 5.98,t, 2.67,dd,                   J = 1.0 Hz                                                                           J = 1.0 Hz                                                                           J = 1.0 Hz                                                                           J = 1.0 Hz                                                                           J = 1.0 Hz                                                                           J = 1.0 Hz                                                                            J = 1.0 Hz                                                                            J = 6.5,                                                                      15.0 Hz                                                                       2.81,dd,                                                                      J = 9.0,                                                                      15.0 Hz                 4                                                     3.67,br m               7  5.52,d,                                                                              5.52,d,                                                                              5.50,d,                                                                              5.51,d,                                                                              5.51,d,                                                                              5.56,d, 5.61,d, 5.45,d,                    J = 9.5 Hz                                                                           J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.5 Hz                                                                           J = 11.0                                                                              J = 10.0 Hz             8  6.60,d,                                                                              6.61,d,                                                                              6.61,d,                                                                              6.61,d,                                                                              6.60,d,                                                                              6.83,d, 6.78,d, 6.58,d,                    J = 9.5 Hz                                                                           J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.0 Hz                                                                          J = 10.5 Hz                                                                           J = 11.0                                                                              J = 10.0 Hz            10  3.90,dq,                                                                             4.16,quin,                                                                           4.26,ddq,                                                                            4.24,dq,                                                                             4.27,dq,                                                                             4.32,dq,                                                                              4.69,dq,                                                                              4.49,dq,                   J-9.0, 6.5 Hz                                                                        J = 6.5 Hz                                                                           J = 1.3, 3.5,                                                                        J = 10.5,                                                                            J = 11.0,                                                                            J = 2.5, 7.0 Hz                                                                       J = 3.0, 6.5                                                                          J = 3.5, 7.0 Hz                          6.5 Hz 6.5 Hz 6.0 Hz                                        11  1.91,ddq,                                                                            2.09,ddd,                                                                            2.23,ddq,                                                                            1.73,ddq,                                                                            1.69,ddq,                                                                            2.22,ddq,                                                                             2.61,dq,                                                                              2.52,dq,                   J = 9.0, 8.0,                                                                        J = 6.0, 6.5,                                                                        J = 3.5,                                                                             J = 10.5,                                                                            J = 11.0, 2.5,                                                                       J = 2.5, 6.0, 7.0                                                                     J = 3.0, 7.0                                                                          J = 3.5, 6.5 Hz            6.5 Hz 7.5 Hz 3.7, 7.5 Hz                                                                          3.3, 6.5 Hz                                                                          6.0 Hz Hz                                     12  4.70,d,                                                                              5.97,d,                                                                              4.31,dd,                                                                             4.95,d,                                                                              4.54,d,                                                                              5.06,dd,                                   J = 8.0 Hz                                                                           J = 6.0 Hz                                                                           J = 3.7,                                                                             J = 3.3 Hz                                                                           J = 2.5 Hz                                                                           J = 6.0, 1.5 Hz                                          1.3 Hz                                                      13,13'                                                                            2.87,m 2.86,m 2.80,m 2.87,m 2.88,m 2.88,m  2.85,m  1.50,m                                   2.92,m               2.79,m          1.80,m                 14,14'                                                                            1.63,m 1.63,sext,                                                                           1.63,m 1.63,sext,                                                                           1.64,sext,                                                                           1.60,sext,                                                                            1.63,sext,                                                                            1.14,m(2H)                        J = 7.0 Hz    J = 7.0 Hz                                                                           J = 7.0 Hz                                                                           J = 7.0 Hz                                                                            J = 7.0 Hz                     15  1.01,t,                                                                              1.01,t,                                                                              1.01,t,                                                                              1.01,t,                                                                              1.01,t,                                                                              0.98,t, 1.01,t, 0.84,t,                    J = 7.5 Hz                                                                           J = 7.0 Hz                                                                           J = 7.5 Hz                                                                           J = 7.5 Hz                                                                           J = 7.5 Hz                                                                           J = 7.5 Hz                                                                            J = 7.5 Hz                                                                            J = 7.0 Hz             16  1.44,s 1.46,s 1.47,s 1.46,s 1.46,s 1.46,s  1.50,s.sup.b                                                                          1.41,s                 17  1.49,s 1.49,s 1.45,s 1.47,s 1.47,s 1.46,s  1.50,s.sup.b                                                                          1.45,s                 18  1.44,d,                                                                              1.43,d,                                                                              1.45,d,                                                                              1.41,d,                                                                              1.38,d,                                                                              1.41,d, 1.42,d, 1.35,d,                    J = 6.5 Hz                                                                           J = 6.5 Hz                                                                           J = 7.0 Hz                                                                           J = 6.0 Hz                                                                           J = 6.5 Hz                                                                           J = 7.0 Hz                                                                            J = 7.0 Hz                                                                            J = 7.0 Hz             19  1.13,d,                                                                              1.05,d,                                                                              1.00,d,                                                                              1.12,d,                                                                              1.13,d,                                                                              1.06,d, 1.14,d, 1.12,d,                    J = 6.5 Hz                                                                           J = 7.5 Hz                                                                           J = 7.5 Hz                                                                           J = 7.0 Hz                                                                           J = 6.5 Hz                                                                           J = 7.5 Hz                                                                            J = 7.5 Hz                                                                            J = 6.5 Hz             OR  3.55,brs (OH)                                                                        2.10,s (OAc)                                                                         3.59,s (OMe)                                                                         2.43,brs (OH)                                                                        3.58,s (OMe)                                                                         3.64,d,         12.4,s (OH)                                                   J = 1.5                                                                       Hz (OH)                                __________________________________________________________________________     .sup.a Specta obtained in CDCl.sub.3. Compounds 1, 7 and 8 were assigned      by HMQC, HMBC, COSY and difference nOe experiments. Assignments for           compounds 2-6 were made by analogy.                                           .sup.b Two peaks separated by 0.04 Hz                                    

                                      TABLE 2                                     __________________________________________________________________________    125 MHz .sup.13 C NMR Data for Compounds 1-8.sup.a                            CARBON                                                                        #     1  2   3   4   5   6   7  8                                             __________________________________________________________________________     2    160.4                                                                            160.0                                                                             160.9                                                                             160.9                                                                             160.8                                                                             160.8                                                                             160.0                                                                            178.6                                          3    110.1                                                                            110.9                                                                             110.7                                                                             110.3                                                                             110.3                                                                             111.1                                                                             111.4                                                                            38.5                                           4    158.9                                                                            157.7                                                                             158.2                                                                             158.7                                                                             158.5                                                                             158.6                                                                             158.1                                                                            30.5                                           4a   104.0                                                                            101.1.sup.b                                                                       102.7                                                                             103.5                                                                             103.2                                                                             103.5.sup.b                                                                       102.7                                                                            108.9                                          4b   151.1                                                                            151.7                                                                             151.6                                                                             151.4                                                                             151.4                                                                             150.6                                                                             157.6                                                                            157.3                                          6    76.6                                                                             77.8                                                                              77.6                                                                              77.7                                                                              77.6                                                                              78.8                                                                              78.9                                                                             78.2                                           7    126.9                                                                            126.8                                                                             126.6                                                                             126.7                                                                             126.6                                                                             126.9                                                                             128.2                                                                            125.6                                          8    116.5                                                                            116.4                                                                             116.6                                                                             116.5                                                                             116.6                                                                             115.7                                                                             115.0                                                                            115.6                                          8a   106.3                                                                            104.1.sup.b                                                                       104.1                                                                             106.1.sup.b                                                                       104.7.sup.b                                                                       102.9.sup.b                                                                       104.1                                                                            102.6                                          8b   153.1                                                                            152.6                                                                             151.6                                                                             153.9                                                                             153.8                                                                             152.6                                                                             160.0                                                                            160.0.sup.f                                   10    77.7                                                                             76.6                                                                              73.8                                                                              73.0                                                                              73.4                                                                              75.6                                                                              77.4                                                                             76.1                                          11    40.5                                                                             38.6.sup.c                                                                        35.1                                                                              38.6.sup.c                                                                        38.66                                                                             35.1                                                                              45.7                                                                             44.2                                          12    67.2                                                                             67.1                                                                              77.6                                                                              61.9                                                                              70.8                                                                              65.9                                                                              192.9                                                                            201.0                                         12a   106.3                                                                            106.2.sup.b                                                                       106.4                                                                             106.2.sup.b                                                                       106.0.sup.b                                                                       109.2.sup.b                                                                       106.8                                                                            101.2                                         12b   154.4                                                                            154.4                                                                             155.1                                                                             153.1                                                                             153.1                                                                             154.6                                                                             154.3                                                                            160.0.sup.f                                   13    38.7                                                                             38.1.sup.c                                                                        38.6                                                                              38.2.sup.c                                                                        38.65                                                                             38.9                                                                              38.9                                                                             35.4                                          14    23.3                                                                             23.3                                                                              23.3                                                                              23.3                                                                              23.3                                                                              23.2                                                                              23.0                                                                             20.7                                          15    14.0                                                                             14.0                                                                              14.0                                                                              14.0                                                                              14.1                                                                              14.0                                                                              13.9                                                                             14.0                                          16    27.4                                                                             27.8                                                                              27.7                                                                              27.8                                                                              27.8                                                                              28.2                                                                              28.0                                                                             28.1                                          17    28.0                                                                             28.0                                                                              27.9                                                                              27.7                                                                              27.9                                                                              28.4                                                                              28.1                                                                             28.5                                          18    18.9                                                                             19.2                                                                              19.5                                                                              18.9                                                                              19.2                                                                              16.8                                                                              15.9                                                                             16.2                                          19    15.1                                                                             15.3                                                                              17.0                                                                              12.5                                                                              13.3                                                                              7.2 9.0                                                                              9.3                                                    170.7.sup.d                                                                       57.6.sup.e                                                                            59.4.sup.e                                                        21.2.sup.d                                                           __________________________________________________________________________     .sup.a Spectra recorded in CDCl.sub.3 and attached protons determined by      the DEPT pulse sequence.                                                      .sup.b,c Resonances within a column may be interchangeable.                   .sup.d Acetyl resonances.                                                     .sup.e Methoxy resonances.                                                    .sup.f In CD.sub.3 OD these signals appeared as doubled peaks at δ      161.20, 161.16 and δ 161.10, 161.03.                               

Example 4

This example illustrates the antiviral activity of calanolides andrelated compounds from Calophyllum lanigerum Miq. var. austrocoriaceum(T. C. Whitmore) P. F. Stevens. Pure compounds were initially evaluatedfor antiviral activity using an XTT-tetrazolium anti-HIV primaryscreening assay described previously (Boyd, M. R., in AIDS Etiology,Diagnosis, Treatment and Prevention (DeVita V. T., Jr., Hellman S.,Rosenberg S. A., eds.), pp 305-319 (Philadelphia: Lippincott, 1988);Gustafson, K. R., et al., J. Med. Chem., 35, 1978-1986 (1992); Weislow,O. S., et al., J. Natl. Cancer Inst., 81, 577-586 (1989); Gulakowski, R.J., et al., J. Virol. Methods, 33, 87-100 (1991)). The CEM-SS humanlymphocytic target cell line used in all assays was maintained in RPMI1640 medium (Gibco, Grand Island, N.Y.), without phenol red, and wassupplemented with 5% fetal bovine serum, 2 mM L-glutamine and 50 μg/mlgentamicin (complete medium). Exponentially-growing cells were pelletedand resuspended at a concentration of 2.0×10⁵ cells/ml in completemedium. The Haitian variant of HIV, HTLV-III_(RF) (3.54×10⁶ SFU/ml), wasused throughout. Frozen virus stock solutions were thawed immediatelybefore use and resuspended in complete medium to yield 1.2×12⁵ SFU/ml.The appropriate amounts of the pure compounds for anti-HIV evaluationswere dissolved in 100% dimethylsulfoxide (DMSO), then diluted incomplete medium to the desired initial concentration (and with the finalDMSO content not exceeding 1%). All serial drug dilutions, reagentadditions, and plate-to-plate transfers were carried out with anautomated Biomek 1000 Workstation (Beckman Instruments, Palo Alto,Calif.).

Over a broad concentration range (<0.1->10 μM), calanolide A (1)provided complete protection against the cytopathic effects of HIV-1infection in the primary screening assay and essentially halted HIV-1reproduction in human T-lymphoblastic (CEM-SS) cells. Calanolide B (4),the C12 epimer of compound 1, also provided complete inhibition of HIV-1in this assay, giving an EC₅₀ =0.4 μM and an IC₅₀ =15.0 μM. The esterderivative, 12-acetoxycalanolide A (2) also was active against HIV-1,albeit somewhat less potent (EC₅₀ =2.7 μM, IC₅₀ =13 μM). Compound 6showed weak but detectable anti-HIV activity, as did the12-methoxycalanolide S (5); however, the antiviral activity of thecorresponding 12-methoxycalanolide A (3) was not detectable in theprimary screen. Compounds 7 and 8 were inactive in the primary screeningassay, indicating the requirement for a fully reduced oxygenfunctionality, or derivative substitutent thereof, at C-12, and thefurther requirement for the intact, characteristic central carbonskeleton shared by compounds 1-6.

For a further demonstration of the anti-HIV activity of pure compounds,a battery of interrelated assays was performed on individual wells of96-well microtiter plates as described in detail elsewhere (Gulakowski,R. J. et al., J. Virol. Methods, 33, 87-100 (1991)). Briefly, theprocedure was as follows. Uninfected CEM-SS cells were plated at adensity of 1×10⁴ cells in 50 μl of complete medium. Diluted HIV-1 viruswas then added to appropriate wells in a volume of 50 μl to yield amultiplicity of infection of 0.6. Appropriate cell, virus and drugcontrols were incorporated in each experiment; the final volume in eachmicrotiter well was 200 μl. Quadruplicate wells were used forvirus-infected cells, and duplicate wells were used for uninfectedcells. Plates were incubated at 37° C. in an atmosphere containing 5%CO₂ for 6 days. Subsequently, aliquots of cell-free supernatant wereremoved from each well using the Biomek, and analyzed for reversetranscriptase activity, p24 antigen production and synthesis ofinfectious virions as described (Gulakowski, R. J., et al., J. Virol.Methods, 33, 87-100 (1991)). Cellular growth or viability then wasestimated on the remaining contents of each well using the XTT (Weislow,O. S., et al., J. Natl. Cancer Inst., 81, 577-586 (1989)), BCECF (Rink,T. L., et al., J. Cell. Biol., 95, 189-196 (1982)) and DAPI (McCaffrey,T. A., et al., In Vitro Cell Develop. Biol., 24, 247-252 (1988)) assaysas described (Gulakowski, R. J., et al., J. Virol. Methods, 33, 87-100(1991)). To facilitate graphical displays and comparisons of data, theindividual experimental assay results (of at least quadruplicatedeterminations of each) were averaged, and the mean values were used tocalculate percentages in reference to the appropriate controls. Standarderrors of the mean values used in these calculations typically averagedless than 10% of the respective mean values.

As illustrated in FIGS. 4A-D, calanolide A was capable of completeinhibition of the cytopathic effects of HIV-1 upon CEM-SS humanlymphoblastoid target cells in vitro (EC₅₀.sup.˜ 0.1 μM); directcytotoxicity of the compound upon the target cells was apparent only at100-fold greater concentrations (IC₅₀.sup.˜ 13 μM; in vitro "therapeuticindex" ≧130). Calanolide A also strikingly inhibited the production ofRT, p24, and SFU in HIV-1-infected CEM-SS within these same inhibitoryeffective concentrations, indicating a cessation of viral replication.Similar results (data not shown) as those depicted for calanolide A inFIGS. 4A-D were also obtained with calanolide B, with a noncytotoxicconcentration of the latter completely inhibiting HIV-replication andaffording complete protection against HIV cytopathicity.

Example 5

This example illustrates antiviral calanolide derivatives and relatedantiviral compounds. One skilled in the art will appreciate that otherantiviral calanolides or antiviral derivatives thereof, in addition tothe calanolides and derivatives of Example 3, may be isolated fromnatural sources and/or be synthesized chemically. Antiviral calanolidesor antiviral derivatives thereof can comprise two distinct series, asillustrated more generally in FIG. 5. For example, calanolide A (1) fromExample 3 is of series 1, wherein R¹ is CH₂ CH₂ CH₃, R² is OH, R⁴ isCH₃, and R⁵ is CH₃. Calanolide B (4) from Example 3 is of series 1,wherein R¹ is CH₂ CH₂ CH₃, R² is OH, R⁴ is CH₃, and R⁵ is CH₃. Compound2 from Example 3 is of series 1, wherein R¹ is CH₂ CH₂ CH₃, R² is O₂CR³, R³ is --CH₃, R⁴ is CH₃, and R⁵ is CH₃. Compound 3 from Example 3 isof series 1 wherein R¹ is CH₂ CH₂ CH₃, R² is OR³, R³ is --CH₃, R⁴ isCH₃, and R⁵ is CH₃. Compound 5 from Example 3 is of series 1, wherein R¹is CH₂ CH₂ CH₃, R² is OR³, R³ is --CH₃, R⁴ is CH₃, and R⁵ is CH₃.Compound 6 from Example 3 is of series 1, wherein R¹ is CH₂ CH₂ CH₃, R²is OH, and R⁴ and R⁵ are each CH₃. The previously known,naturally-occurring compound (Stout, G. H., and Stevens, K. L., J. Org.Chem., 29, 3604-3609 (1964)) costatolide (compound 9 of FIG. 2) is anexample of series 1, wherein R¹ is CH₂ CH₂ CH₃, R² is OH, R⁴ is CH₃, andR⁵ is CH₃. Compound 7 from Example 3 illustrates a related naturalproduct from Calophyllum lanigerum which has the same carbon skeletonthat characterizes members of series 1, wherein R¹ is CH₂ CH₂ CH₃ and R⁴and R⁵ are each CH₃.

Whereas the above examples provide the essential precedents for thecharacteristic structural (stereochemical and substituent) featuresabout C-10, C-11, and C-12 relative to the antiviral calanolides andantiviral derivatives thereof of FIG. 5, there are also precedents forvariations at R¹ provided by related naturally occurring compoundshaving variations at C-4. For example, compounds, which have the samecentral carbon skeleton that characterize the antiviral calanolides andantiviral derivatives thereof of FIG. 5, have been isolated that haveCH₃ or aryl substituents at C-4 (Dharmaratne, H. R. W., et al.,Phytochemistry, 24, 1553-1556 (1985); Gunasekera, S. P., et al., J.Chem. Soc. Perkin I, 1505-1511 (1977)), but differ in thestereochemistry and/or substituents at C-10, C-11, and C-12 thatcharacterize the calanolides. For example, the previously known(Gunasekera, S. P., et al., J. Chem. Soc. Perkin I, 1505-1511 (1977)),naturally occurring compound soulattrolide (compound 14 of FIG. 2) is anexample of a series 1 compound, wherein R¹ is C₆ H₅, R² is OH, R⁴ isCH₃, and R⁵ is CH₃.

As a more specific illustration, the isolation and purification ofcostatolide (9) and soulattrolide (14) from latex of Calophyllumteysmannii var. inophylloide is provided in Example 8 below. Thepreviously unknown antiviral activity of these two compounds is shown inExample 10 and Table 6 below.

Whereas the above examples provide precedents for the isolation ofnaturally occurring antiviral calanolides and antiviral derivativesthereof, and of other natural products that show the essentialstructural features of antiviral calanolides and derivatives thereof ofseries 1, one skilled in the art also will appreciate that, usingstandard organic chemical methodology, a number of structuralmodifications can be made for purposes of preparing antiviralcalanolides or antiviral derivatives thereof. For example, antiviralcalanolides and antiviral derivatives of series 2 can be made fromcalanolides or derivatives thereof of series 1. More specifically, anantiviral member of series 1 may be converted to the correspondingmember of series 2, wherein C7 and C8 of the latter are fully saturated.7,8-dihydro calanolide A of series 2 can be made from calanolide A ofseries 1 by platinum oxide-catalyzed hydrogenation of the 7,8 olefiniclinkage.

Example 9 below more specifically illustrates the conversion of series 1compounds (calanolide A, calanolide B, costatolide, and soulattrolide)to series 2 compounds (7,8-dihydrocalanolide A, 7,8-dihydrocalanolide B,7,8-dihydrocostatolide, and 7,8-dihydrosoulattrolide, respectively).

As a further example, an antiviral member of either series 1 or 2,wherein R² is OH or OH can be converted to the corresponding C-12 esteror sulfonate ester wherein R² is O₂ CR³, O₂ CR³, O₃ SR³, or O₃ SR³, byreaction with the corresponding acid halide, X--OCR³, or X--O₂ SR³,where X═Cl, Br, or I, and R³ is C₁ -C₆ alkyl or aryl, or, alternatively,by reaction directly with the corresponding acids, HO₂ CR³, or HO₃ SR³,wherein R³ is C₁ -C₆ alkyl or aryl, and dicyclohexylcarbodiimide inanhydrous pyridine or triethylamine. Vice versa, a C-12 ester of eitherseries 1 or 2, wherein R² is O₂ CR³, O₂ CR³, O₃ SR³ or O₃ SR³, and R³ isC₁ -C₆ alkyl or aryl, may be hydrolyzed chemically or enzymatically tothe respective parent calanolide compound, wherein R² is OH or OH. It isfurther noted that calanolide derivatives esterified at C-12, which aresusceptible to plasma- or tissue-esterase mediated deesterification, canserve as prodrugs in vivo for antiviral calanolides, wherein the C-12substituent is OH or OH.

Example 6

This example sets forth the taxonomic characteristics of Calophyllumteysmannii var. inophylloide (Soejarto et al. 7853, 7854, 7899, 7900,7901, 7902) used in Examples 7 and 8. Of the five trees with dbh>30 cm,tree 7854 grows on a ridge, while 7899-7902 are found along gentleslopes, towards streams found at lower sides of these slopes, in a deepkerangas forest at Sampedi Forest Reserve, which has been selectivelylogged. These 5 trees are situated along a more-or-less straight line,each tree separated from the other by a distance of between 40 and 75 m;the distance between trees 7854 and 7902 (at the opposite ends of thestraight line) is about 300 m. Further search indicated that within this0.3 hectare of this forest, only those 5 large trees of Calophyllumteysmannii var. inophylloide were present. Based on this observation, itwas estimated that approximately 15 large (dbh>30 cm) trees might beexpected to be found in a one-hectare forest in this locality. However,many more small trees of this species (saplings and seedlings) werefound.

These five trees are characterized taxonomically as follows: tree 12-25m tall, dbh 30-60 cm, trunk at base with low spurs, bark gray-brown togray-black, roughly fissured, cracked, and scaling off, slash lightpinkish red to pinkish brown, latex clear-yellow, seeping out as tinydroplets or en masse and collecting on the cut surface of the bark,sticky; twigs slightly flattened-to-angled, covered with blackish brownpubescence, terminal buds small, somewhat conical and flattened, 1 cmlong, covered by similar pubescence as the twig; leaves stronglycoriaceous, dark olive-brown above, paler beneath, narrowly obovate toelliptic-obovate-to-oblong, 10-18 cm long, 3-6 cm wide, apex rounded,base cuneate to rounded-cuneate, margins entire sharply recurved, veins14-16 per 5 mm, petiole 1.5-2 cm long, canaliculate. All trees weresterile at the time of collection. The characteristic features ofCalophyllum teysmannii var. inophylloide as compared to Calophyllumlanigerum var. austrocoriaceum are summarized in Table 3. According toStevens, P. F., J. Arnold Arbor., 61, 356-361, 431-443 (1980), sterilespecimens of C. teysmannii var. inophylloide are distinguished from C.lanigerum var. austrocoriaceum by the larger tree size, the dark brownto blackish, rough and coarsely fissured and cracked trunkbark (vs. thelighter colored, smoothish and lenticellate bark of C. langerium var.austrocoriaceum), the smaller and less plump terminal buds, the normallyless elongate leaves which are more strongly coriaceous in texture, andthe recurved leaf margins.

                                      TABLE 3                                     __________________________________________________________________________    Distinguishing features between Calophyllum teysmannii var. inophylloide      and Calophyllum lanigerum var. austrocoriaceum                                __________________________________________________________________________    Tree   to 40 m tall, dbh to 95 cm                                                                    to 21 m tall, dbh to 48 cm                             Bark   gray-black, roughly fissured and                                                              brownish yellow to dark brown,                                cracked         smoothish and lenticellate, either                                            irregularly arranged in a ring                                                or in longitudinal rows                                Trunkbase                                                                            with spurs/small buttresses at                                                                no spur or buttresse                                          base                                                                   Leaf blade                                                                           elongate ovate to elliptic to                                                                 obovate to narrowly ovate to                                  oblong 7-17 cm long by 3-5 cm                                                                 oblong, 4-20 cm long by 3-8 cm                                wide, strongly coriaceous                                                                     wide, coriaceous margins not                                  margins recurved                                                                              recurved                                               Lateral veins                                                                        6-21 per 5 mm   6-15 per mm                                            Terminal bud                                                                         small, 0.2-1 cm long                                                                          large and plump, 1.5-3 cm long                         __________________________________________________________________________

Example 7

This example illustrates the collection of latex (resinous exudate) fromthe Calophyllum teysmannii var. inophylloide trees of Example 6(Soejarto et al. 7853, 7854, 7899, 7900, 7901, 7902). On the trunkbarkat breast-high of tree 7854, two old wounds (slashes) made on Jul. 19,1992, had healed by the visit of Jan. 7, 1993. On these old wounds,latex crusts that had dried up from the July 1992 cuts remained. Theselatex crusts were of opaque, light yellowish color. These were collectedby scraping the dried crusts with a pocket knife and placing them in asmall polyethylene (plastic) bag. In addition to these old wounds, newslashes were made, from which a fresh latex sample was collected andplaced in a separate plastic bag. All latex samples were numberedaccordingly.

Slashes were also made on trees 7899-7902, and latex samples scrapedfrom the cut surfaces, where enough quantity would collect within 5-20minutes after a slash was made. Since the latex is scanty and is notfree-flowing, the term "tapping" is not appropriate for collecting latexsamples of Calophyllum species. The term "scraping," such as by using apocket knife, is more appropriate.

Slashing method: clean slashes (cuts) of varying sizes, 3-25 cm long by1.5-3 cm wide, were made using a large cutting tool (a machete or"parang"), taking care not to cut the cambium layer. From 4 to 9 slashesper tree at breast high were made. Direction of slashes ranges fromhorizontal (perpendicular to the tree axis) to a slant of 45° angle.After enough latex has accumulated on the slashed surfaces, this isscraped carefully using a pocket knife.

Scraping method: a pocket knife is held and traced along the contour ofthe slash/cut to gather the latex. (a) One scraping operation was madefrom all the slashes on every tree in late morning of Jan. 7, 1993; thetotal latex sample from all slashes on one tree was placed in a smallplastic bag. (b) A second scraping was made on all slashes of every treein the late afternoon of Jan. 7, 1993; the total latex sample from anumber of slashes on one tree was placed in a small plastic bag,separate from the morning scraping. (c) A third scraping was made on allslashes of every tree in the morning of Jan. 8, 1993; the total latexfrom a number of slashes on one tree was placed in a small plastic bag,separate from those made on Jan. 7, 1993. (d) A fourth scraping was madeon all slashes of every tree on the week of Jan. 11, 1993; the totallatex from a number of slashes on one tree was placed in a small plasticbag, separate from those made previously.

It was noticed that all latex samples changed in appearance (color) fromclear-yellow (upon exudating on the cut surface) to opaque-yellow,following scraping and handling in the plastic bag. When dry (in thebag), it became "crumbly" to more-or-less "sandy" in texture. One dayafter collection, latex samples in the plastic bag emitted a ratherstrong, characteristic odor, which is not unpleasant.

These samples were kept in a dry place at all times and were packed intoseveral larger plastic bags. These bags were shipped by air to the U.S.as carry-on luggage, without any special treatment or handling, andarrived on Jan. 15, 1993 in good condition; each bag continued to emitthe same characteristic odor. A summary of latex yields from the fivetrees is presented in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Trunkbark-latex yield of Calophyllum teysmannii var. inophylloide             Collector's #                                                                         Tree       Scraping Day/Time                                          (NCI Barcode)                                                                         DBH # of Slashes                                                                         Jan 7/am                                                                           Jan 7/pm                                                                           Jan 8/am                                                                           Wk Jan 11                                                                           Tot. Yield                            __________________________________________________________________________    Soejarto et al.                                                                       16 cm                                                                             7(3-6 cm × (1.5 g)     (1.5 g)                              7853        11/2-2 cm)                                                        (U44Z-6117-K)                                                                 Soejarto et al.                                                                       37 cm                                                                             4(8-20 cm ×                                                                    2.5 g                                                                              2.4 g                                                                              3.8 g                                                                              10.0 g                                                                              18.7 g                                7854        11/2-3 cm)                                                        (U44Z-6118-L)                                                                 Soejarto et al.                                                                       57 cm                                                                             9(6-15 cm ×                                                                    1.1 g                                                                              3.4 g                                                                              5.0 g                                                                              10.0 g                                                                              19.5 g                                7899        1-4 cm)                                                           (U44Z-6996-P)                                                                 Soejarto et al.                                                                       32 cm                                                                             9(8-13 cm ×                                                                    1.1 g                                                                              2.0 g                                                                              4.4 g                                                                              8.0 g 15.5 g                                7900        1-3 cm)                                                           (U44Z-6997-Q)                                                                 Soejarto et al.                                                                       55 cm                                                                             7(5-15 cm ×                                                                    0.5 g                                                                              2.1 g                                                                              7.7 g                                                                              7.0 g 17.3 g                                7901        2-6 cm)                                                           (U44Z-6998-R)                                                                 Soejarto et al.                                                                       45 cm                                                                             9(8-25 cm ×                                                                    2.6 g                                                                              9.2 g                                                                              2.0 g                                                                              8.5 g 22.3 g                                7902        2-5 cm)                                                           (U44Z-6999-S)                                                                 Total trunkbark-latex yield from 5 trees with DBH >30 cm in 4                                                         93.3 gngs                             __________________________________________________________________________

Example 8

This example sets forth the isolation of costatolide (compound 9 of FIG.2) and soulattrolide (compound 14 of FIG. 2) from the latex of Example7. Neither of these previously known compounds were known to occur inthe latex of Calophyllum teysmannii var. inophylloide nor were theyknown to have antiviral activity.

A crude Calophyllum TEYSMANNII var. inophylloide latex sample (30 g) wastriturated 3 times with 250 ml of CHCl.sub. -MeOH (1:1). The solutionwas filtered and evaporated to give 19.9 g of extract. In a typicalisolation, 100 mg of resin extract was separated by SiO₂ HPLC (41.1×300mm, Dynamax column) eluted with 75 ml/min hexane/EtOAc (7:3), to give 20mg of costatolide (retention time=8.5 min; [α]_(D) =-19.7 (CHCl₃, c1.1); EIMS m/z 370, appropriate for C₂₂ H₂₆ O₅ ; ¹ H MNR, see Table 5 inExample 9 below), and 13 mg of soulattrolide (retention time=9.6 min;[α]_(D) =-25.0 (CHCl₃, c 0.9), EIMS m/z 404, appropriate for C₂₅ H₂₄ O₅; ¹ H NMR, see Table 5 in Example 9 below).

Example 9

This example illustrates the preparation of antiviral 7,8-dihydrocompounds (FIG. 6) of series 2, specifically 7,8-dihydrocalanolide A(FIG. 6A), 7,8-dihydrocalanolide B (FIG. 6B), 7,8-dihydrocostatolide(FIG. 6), and 7,8-dihydrosoulattrolide (FIG. 6D) from the corresponding7,8-unsaturated compounds of series 1. Of these six compounds, only onestructure (7,8-dihydrocostatolide (FIG. 6C); Stout, G. H., et al., J.Org. Chem., 29, 3604-3609 (1964)) had been previously reported, howevernone of the six were known heretofore to have antiviral activity. The7,8-dihydro compounds were made by catalytic hydrogenation of theselected appropriate 7,8-unsaturated precursors. In a typical procedure,10 mg of calanolide A was stirred with 2.5 mg PtO₂ (Aldrich) in 4 mlMeOH under an atmosphere of H₂ (a balloon filled with H₂ was attached tothe reaction vessel to provide a slightly positive pressure of H₂) for30 min. The mixture was filtered, and the solvent removed in vacuo toprovide a light yellow oil which was purified by SiO₂ HPLC (21.1×300 mm,Dynamax column) eluted with 25 ml/min hexane/EtOAc (7:3) to give 9.0 mgof 7,8-dihydrocalanolide A (EIMS m/z 372, appropriate for C₂₂ H₂₈ O₅ ; ¹H NMR, see Table 5). In an exactly analogous fashion, 6 mg of calanolideB with 3 mg PtO₂ in 4 ml MeOH was converted to 3.6 mg of7,8-dihydrocalanolide B (EIMS m/z 372, appropriate for C₂₂ H₂₈ O₅ ; ¹ HNMR, see Table 5); 100 mg of costatolide with 15 mg PtO₂ in 20 ml MeOHwas converted to 89.9 mg 7,8-dihydrocostatolide (EIMS m/z 372,appropriate for C₂₂ H₂₈ O₅ ; ¹ H NMR, see Table 5); and 12 mg ofsoulattrolide with 3 mg PtO₂ in 4 ml MeOH was converted to 8.9 mg7,8-dihydrosoulattrolide (EIMS m/z 406, appropriate for C₂₅ H₂₆ O₅ ; ¹ HNMR, see Table 5).

                                      TABLE 5                                     __________________________________________________________________________    500 MHz NMR Data for Costatolide, Soulattrolide,                              7,8-dihydrocalanolide A, 7,8-dihydrocalanolide B, 7,8-dihydrocostatolide,     and 7,8-dihydrosoulattrolide                                                  Proton                                              7,8-                      #   Costatolide                                                                         Soulattrolide                                                                        7,8-Dihydrocalanolide A                                                                   7,8-Dihydrocalanolide B                                                                   7,8-Dihydrocostatolide                                                                   Dihydrosoulattrolide      __________________________________________________________________________     3  5.93 t                                                                              5.94 s 5.90 s      5.90 s      5.90 s     5.92 s                        J = 1.0 Hz                                                                 7  5.51 d                                                                              5.34 d 1.75 (2H) m 1.76 (2H) t 1.76 (2H) t                                                                              1.55 (2H) t                   J = 10.0                                                                            J = 9.8 Hz         J = 6.8 Hz  J = 6.8 Hz J = 6.8                       Hz                                                                         8  6.61 d                                                                              6.51 d 2.61 (2H) m 2.61 (2H) m 2.61 (2H) m                                                                              2.53 (2H) m                   J = 10.0                                                                            J = 9.8 Hz                                                              Hz                                                                        10  4.24 dq                                                                             4.26 dq                                                                              3.89 dq     4.22 dq     4.22 dq    4.25 dq                       J = 10.5;                                                                           J = 10.7; 6.4                                                                        J = 8.8; 6.4 Hz                                                                           J = 10.7; 6.3 Hz                                                                          J = 10.7; 6.3 Hz                                                                         J = 10.7; 6.4 Hz              6.5 Hz                                                                              Hz                                                                  11  1.73 ddq                                                                            1.76 m 1.88 m      1.72 m      1.72 m     1.75 m                        J = 10.5;                                                                     3.3; 6.5 Hz                                                               12  4.95 d                                                                              5.02 d 4.71 d      4.97 d      4.97 d     5.04 d                        J = 3.3 Hz                                                                          J = 3.4 Hz                                                                           J = 7.8 Hz  J = 2.9 Hz  J = 2.9 Hz J = 2.9                   13  2.87 m       2.83 m; 2.90 m                                                                            2.84 m; 2.89 m                                                                            2.84 m; 2.89 m                       14  1.63 sext                                                                           7.22 (2H) m                                                                          1.60 m      1.60 sext   1.60 sext  7.19 (2H) m                   J = 7.0 Hz               J = 7.3 Hz  J = 7.3 Hz                           15  1.01 t                                                                              7.35 (3H) m                                                                          0.99 t      0.99 t      0.99 t     7.33 (3H) m                   J = 7.5 Hz   J = 7.4     J = 7.3     J = 7.3                              16  1.46 s                                                                              0.91 s 1.33 s      1.35 s      1.35 s     0.80 s                    17  1.47 s                                                                              0.91 s 1.37 s      1.35 s      1.35 s     0.81 s                    18  1.41 d                                                                              1.14 d 1.43 d      1.40 d      1.35 s     1.41 d                        J = 6.0                                                                             J = 6.3 Hz                                                                           J = 6.4     J = 5.9     J = 5.9    J = 6.4 Hz                19  1.12 d                                                                              1.14 d 1.12 d      1.12 d      1.12 d     1.14 d                        J = 7.0 Hz                                                                          J = 6.8                                                                              J = 6.8     J = 7.3     J = 7.3 Hz J = 6.8                   __________________________________________________________________________                                                        Hz                    

Example 10

This example further illustrates the antiviral activities of thecalanolides, related compounds, and derivatives thereof. Morespecifically, FIG. 7 shows comparative anti-HIV profiles of calanolide A(FIG. 7A), calanolide B (FIG. 7B), costatolide (FIG. 7C), soulattrolide(FIG. 7D), and the four corresponding 7,8-dihydro reduction products(FIG. 7E-H). The profiles were obtained using the XTT anti-HIV-1 assay(described above in Example 4) on all eight compounds testedside-by-side in the same week. In this initial comparison, clearly alleight compounds had strong anti-HIV activity, indeed capable ofcompletely protecting the CEM-SS target cells from the killing effectsof HIV-1. However, there did appear to be some modest differences inantiviral potencies (e.g., EC_(50's)) and/or direct cytotoxicities(e.g., IC_(50's)) to the target cells.

More detailed, quantitative comparison of four of the above compoundswas obtained by quadruplicate tests of each compound using two differentanti-HIV assay methods, the XTT-based assay as well as the BCECF-basedassay, both as described in Example 4 above. The results are summarizedin Table 6. Overall, with this particular HIV-1 strain (RF) and hostcell line (CEM-SS), calanolide A most consistently gave the highestantiviral potency (lowest EC₅₀); 7,8-dihydrocalanolide A and costatolidewere approximately equipotent and about 2/3 the potency of calanolide A;7,8-dihydrocostatolide was about 1/3 as potent as calanolide A. Thedirect cytotoxicity of the compounds to the CEM-SS target cells wasgreatest (lowest IC₅₀) for costatolide; however, it was somewhat lessfor 7,8-dihydrocostatolide, which in turn was more comparable tocalanolide A and 7,8-dihydrocalanolide A. The in vitro "therapeuticindex" (TI) was consistently greatest for calanolide A and7,8-dihydrocalanolide A.

                                      TABLE 6                                     __________________________________________________________________________    Comparison of antiviral activities (EC.sub.50's) and cytotoxicities           (IC.sub.50's) of calanolide A,                                                7,8-dihydro(DH)calanolide A, costatolide, and 7,8-dihydro(DH)costatolide      from                                                                          quadruplicate tests of each compound with XTT-based and                       BCECF-based anti-HIV-1 assays                                                          EC.sub.50 μM (±SD)                                                                  IC.sub.50 μM (±SD)                                                                  TI (IC.sub.50 /EC.sub.50)                    Compound XXT   BCECF XTT   BCECF XTT                                                                              BCECF                                     __________________________________________________________________________    Calanolide A                                                                           0.13 (0.01)                                                                         0.16 (0.09)                                                                         14.3 (1.65)                                                                         14.3 (1.83)                                                                         110                                                                              89                                        DH-calanolide A                                                                        0.23 (0.03)                                                                         0.19 (0.07)                                                                         12.5 (1.12)                                                                         18.7 (1.95)                                                                         54 98                                        Costatolide                                                                            0.23 (0.04)                                                                         0.20 (0.07)                                                                         10.0 (0.41)                                                                          8.0 (0.97)                                                                         43 40                                        DH-costatolide                                                                         0.30 (0.01)                                                                         0.31 (0.06)                                                                         18.7 (1.33)                                                                         10.3 (3.06)                                                                         62 33                                        __________________________________________________________________________

Quadruplicate tests of each of the above four compounds on HIV-1reproductive indices (also assayed as described in Example 4 above)yielded the results set forth in Table 7, which are consistent with theresults and conclusions from Table 6.

                  TABLE 7                                                         ______________________________________                                        Comparison of in vitro suppression of supernatant viral                       reproductive indices (viral core protein [p24]; reverse                       transcriptase [RT], and syncytium-forming units [SFU])                        by calanolide A, 7,8-dihydrocalanolide A, costatolide, and                    7,8-dihydrocostatolide                                                                  EC.sub.50 μM (±S.D.)                                          Compound    p24        RT         SFU                                         ______________________________________                                        Calanolide A                                                                              0.07 (0.01)                                                                              0.07 (0.01)                                                                              0.08 (0.02)                                 DH-calanolide A                                                                           0.12 (0.02)                                                                              0.13 (0.03)                                                                              0.17 (0.03)                                 Costatolide 0.11 (0.03)                                                                              0.12 (0.04)                                                                              0.14 (0.07)                                 DH-costatolide                                                                            0.24 (0.04)                                                                              0.25 (0.30)                                                                              0.19 (0.11)                                 ______________________________________                                    

All references identified herein (including publications, patents,patent applications, and the like) are hereby incorporated by referencein their entireties.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred compounds, compositions, and methods may be varied.It is intended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the followingclaims.

What is claimed is:
 1. A method of treating a HIV-1 infection, whichmethod comprises administering to a human an anti-HIV-1 effective amountof at least one compound selected from the group consisting of: ##STR3##wherein R¹ is C₁ -C₆ alkyl or aryl; R² is OH, OR³, OR³, O₂ CR³, O₂ CR³,O₃ SR³, or OH, O₃ SR³ ; wherein R³ is C₁ -C₆ alkyl or aryl; and R⁴ andR⁵ are the same or different and are each CH₃ or CH₃.
 2. The method ofclaim 1, which further comprises co-administering an anti-HIV-1effective amount of at least one additional antiviral compound otherthan said compound.
 3. The method of claim 1, wherein R³ is C₁ -C₆ alkylor phenyl.
 4. The method of claim 3, which further comprisesco-administering an anti-HIV-1 effective amount of at least oneadditional antiviral compound other than said compound.
 5. The method ofclaim 3, wherein said compound is selected from the group consisting ofcalanolide A, dihydrocalanolide A, calanolide B, and dihydrocalanolideB.
 6. The method of claim 5, which further comprises co-administering ananti-HIV-1 effective amount of at least one additional antiviralcompound other than said compound.
 7. A compound, in substantially pureform, selected from the group consisting of calanolide A,dihydrocalanolide A, calanolide B, dihydrocalanolide B, anddihydrosoulattrolide.
 8. The compound of claim 7, in substantially pureform, which is calanolide A.
 9. The compound of claim 7, insubstantially pure form, which is dihydrocalanolide A.
 10. The compoundof claim 7, in substantially pure form, which is calanolide B.
 11. Thecompound of claim 7, in substantially pure form, which isdihydrocalanolide B.
 12. The compound of claim 7, in substantially pureform, which is dihydrosoulattrolide.
 13. A method of isolating anantiretroviral compound of claim 1 from plants of the genus Calophyllum,which method comprises the step of:(a) extracting plant material with anorganic solvent to obtain a crude extract having antiretroviralactivity; (b) solvent-solvent partitioning said crude extract asnecessary to concentrate an antiretroviral compound in a nonpolarfraction; (c) subjecting said crude extract or said nonpolar fraction togel permeation or vacuum liquid chromatography as necessary to furtherconcentrate said antiretroviral compound; and (d) isolating andpurifying said antiretroviral compound by HPLC on silica gel and C₁₈reversed-phase columns.
 14. The method of claim 13, wherein said plantmaterial consists of leaves, stems, twigs, fruits, flowers, wood, barkor roots of the said Calophyllum plants.
 15. The method of claim 13,wherein said plant material consists of latex from the said Calophyllumplants.
 16. The method of claim 15, wherein said latex is obtainednon-destructively from Calophyllum plants by the steps of:(a) makingmultiple superficial slashes to the trunkbark, extending to but notthrough the cambium layer; and (b) periodically scraping the accumulatedlatex from the slashed surfaces.
 17. The method of claim 13, whereinsaid plant material is from Calophyllum lanigerum Miq.
 18. The method ofclaim 17, wherein said plant material is from Calophyllum lanigerum Miq.var. austrocoriaceum (T. C. Whitmore) P. F. Stevens.
 19. The method ofclaim 13, wherein said plant material is from Calophyllum teysmanniiMiq.
 20. The method of claim 19, wherein said plant material is fromCalophyllum teysmannii Miq. var. inophylloide (King) P. F. Stevens. 21.The method of claim 20 wherein said plant material is latex and saidantiretroviral compound is costatolide or soulattrolide.
 22. Apharmaceutical composition which comprises a pharmaceutically acceptablecarrier and an anti-HIV-1 effective amount of at least one compoundselected from the group consisting of calanolide A, dihydrocalanolide A,calanolide B, dihydrocalanolide B, and dihydrosoulattrolide.
 23. Thecomposition of claim 22, wherein said compound is selected from thegroup consisting of calanolide A, dihydrocalanolide A, calanolide B, anddihydrocalanolide B.
 24. The composition of claim 22, which furthercomprises an anti-HIV-1 effective amount of at least one additionalanti-HIV-1 compound other than a compound selected from the groupconsisting of calanolide A, dihydrocalanolide A, calanolide B,dihydrocalanolide B, costatolide, dihydrocostatolide, soulattrolide, anddihydrosoulattrolide.